Novel ligand and dna thereof

ABSTRACT

Peptides (I) (of six sequences (GPR7 ligands A through F) of human, mouse, rat and bovine origin, given in the specification) which bind to the GPR7 opioid-somatostatin-like receptor protein, and the amides, esters and salts of these peptides, and their derivatives brominated at the N-terminal amino acid, are new. Independent claims are also included for (1) peptides containing partial sequences of (I); (2) precursor peptides of (I); (3) polynucleotides encoding all or part of (I); (4) expression vectors containing these polynucleotides; (5) hosts transformed by the expression vectors; (6) preparation of (I) or their partial peptides or precursors, by culture of host cells transformed by the expression vectors; (7) antibodies to all or part of (I); (8) drug compositions containing peptides (I), their partial peptides, polynucleotides encoding them, or antibodies recognizing them; (9) diagnostic reagents containing polynucleotides encoding all or part of (I), or antibodies recognizing all or part of (I); (10) screening compounds modifying the binding of (I) to GPR7 or modifying the degree of expression of (I); (11) compounds identified by the screening method; (12) drug compositions containing these compounds; (13) kits for carrying out the screening method; (14) bovine GPR7 receptor protein and its partial peptides; (15) polynucleotides encoding all or part of bovine GPR7; (16) expression vectors containing these polynucleotides; (17) hosts transformed by the expression vectors; (18) preparation of bovine GPR7 and its partial peptides by culture of the transformed host cells; (19) antibodies to bovine GPR7; (20) drug compositions containing bovine GPR7, its partial peptides, polynucleotides encoding it or antibodies recognizing it; (21) diagnostic reagents containing antibodies to bovine GPR7 or polynucleotides encoding all or part of bovine GPR7; (22) bovine GPR8 receptor protein and its partial peptides; (23) polynucleotides encoding all or part of bovine GPR8; expression vectors containing these polynucleotides; (24) hosts transformed by the expression vectors; (25) preparation of bovine GPR8 and its partial peptides by culture of the transformed host cells; (26) antibodies to bovine GPR8; (27) drug compositions containing bovine GPR8, its partial peptides, polynucleotides encoding it or antibodies recognizing it; (28) diagnostic reagents containing antibodies to bovine GPR8 or polynucleotides encoding all or part of bovine GPR8; (29) non-human mammals which are transgenic animals containing foreign DNA encoding (I), bovine GPR7 or bovine GPR8, or are knockout animals for the genes encoding (I), bovine GPR7 or bovine GPR8; (30) non-human mammal embryonic stem cells (ES cells) having the gene for (I), bovine GPR7 or bovine GPR8 in an inactivated form; (31) expression vectors for (I) in mammals; and (32) screening compounds for their ability to modify the promoter activity of the gene for (I), bovine GPR7 or bovine GPR8, using non-human mammals transformed by a reporter gene under the control of the promoter region of the gene for (I), bovine GPR7 or bovine GPR8. ACTIVITY : Anorectic; Cytostatic. No biological data is given. MECHANISM OF ACTION : Modifying the binding of the opioid-somatostatin-like receptor GPR7 to its ligands; (I) and N-terminal brominated derivatives have an agonist effect on GPR7 expression.

FIELD OF THE INVENTION

[0001] The present invention relates to a peptide capable of binding toGPR7, etc., its DNA and use thereof, bovine-derived GPR7 and GPR8, DNAsthereof as well as use thereof.

BACKGROUND ART

[0002] Important biological functions including maintenance ofhomeostasis in the living body, reproduction, development ofindividuals, metabolism, growth, control of the nervous, circulatory,immune, digestive or metabolic system, sensory adaptation, etc. areregulated by cells that receive endogenous factors such as varioushormones and neurotransmitters or sensory stimulation like light orodor, via specific receptors present on cell membranes reserved forthese endogenous factors or stimulation and interact with them. Many ofthese receptors for hormones or neurotransmitters, which take part insuch functional regulation, are coupled to guanine nucleotide-bindingproteins (hereinafter, sometimes merely referred to as G proteins), andare characterized by developing a variety of functions through mediationof intracellular signal transduction via activation of the G proteins.In addition, these receptor proteins possess common seven transmembraneregions. Based on the foregoing, these receptors are thus collectivelyreferred to as G protein-coupled receptors or seven transmembranereceptors. As such, it is known that various hormones orneurotransmitters and their receptor proteins are present and interactwith each other to play important roles for regulating the biologicalfunctions. However, it often remains unclear if there are any otherunknown substances (hormones, neurotransmitters, etc.) and receptors tothese substances.

[0003] In recent years, accumulated sequence information of human genomeDNA or various human tissue-derived cDNAs by random sequencing and rapidprogress in gene analysis technology have been accelerating theinvestigation of human genome. With such advance, it has been clarifiedthat there are many genes supposed to encode proteins with unknownfunctions. G protein-coupled receptors not only have seven transmembranedomains but many common sequences are present in their nucleic acids oramino acids. Thus, these receptors can be precisely identified to be Gprotein-coupled receptors in such proteins. On the other hand, these Gprotein-coupled receptor genes are obtained also by polymerase chainreaction (hereinafter abbreviated as PCR) utilizing such a structuralsimilarity. In these G protein-coupled receptors thus obtained so far,ligands to some receptors that are subtypes having high homology instructure to known receptors may be readily predictable but in mostcases, their endogenous ligands are unpredictable so that ligandscorresponding to these receptors are not found. For this reason, thesereceptors are termed orphan receptors. It is likely that unidentifiedendogenous ligands to such orphan receptors would participate inbiological phenomena poorly analyzed because the ligands were unknown.When such ligands are associated with important physiological effects orpathologic conditions, it is expected that development of these receptoragonists or antagonists will result in breakthrough new drugs (Stadel,J. et al., TiPS, 18, 430-437, 1997; Marchese, A. et al., TiPS, 20,370-375, 1999; Civelli, O. et al., Brain Res., 848, 63-65, 1999, Howard,A. D. et al, TiPS, 22, 132-140, 2001).

[0004] Recently, some groups attempted to investigate ligands to theseorphan receptors and reported isolation/structural determination ofligands, which are novel physiologically active peptides. Independently,Reinsheid et al. and Meunier et al. introduced a cDNA coding for orphanG protein-coupled receptor LC132 or ORL1 into animal cells to express areceptor, isolated a novel peptide from porcine brain or rat brainextract, which was named orphanin FQ or nociceptin, with reference toits response and determined its sequence (Reinsheid, R. K. et al.,Science, 270, 792-794, 1995; Meunier, J.-C. et al., Nature, 377,532-535, 1995). This peptide was reported to be associated with pain.Further research on the receptor in knockout mice reveals that thepeptide takes part in memory (Manabe, T. et al., Nature, 394, 577-581,1998).

[0005] Subsequently, novel peptides such as PrRP (prolactin releasingpeptide), orexin, apelin, ghrelin and GALP (galanin-like peptide), etc.were isolated as ligands to orphan G protein-coupled receptors (Hinuma,S. et al., Nature, 393, 272-276, 1998; Sakurai, T. et al., Cell, 92,573-585, 1998; Tatemoto, K. et al., Biohem. Biophys. Res. Commun., 251,471476, 1998; Kojima, M. et al., Nature, 402, 656-660, 1999; Ohtaki, T.et al., J. Biol. Chem., 274, 37041-37045, 1999). On the other hand, somereceptors to physiologically active peptides, which were hithertounknown, were clarified. It was revealed that a receptor to motilinassociated with contraction of intestinal tracts was GPR38 (Feighner, S.D. et al., Science, 284, 2184-2188, 1999). Furthermore, SLC-1 wasidentified to be a receptor to MCH (Chambers, J. et al., Nature, 400,261-265, 1999; Saito, Y. et al., Nature, 400, 265-269, 1999; Shimomura,Y. et al., Biochem. Biophys. Res. Commun. 261, 622-626, 1999; Lembo, P.M. C. et al., Nature Cell Biol., 1, 267-271, 1999; Bachner, D. et al.,FEBS Lett., 457, 522-524, 1999). Also, GPR14 (SENR) was reported to be areceptor to urotensin II (Ames, R. S. et al., Nature, 401, 282-286,1999; Mori, M. et al., Biochem. Biophys. Res. Commun., 265, 123-129,1999; Nothacker, H.-P. et al., Nature Cell Biol., 1, 383-385, 1999, Liu,Q. et al., Biochem. Biophys. Res. Commun., 266, 174-178, 1999). Besides,receptors to neuromedin U and neuropetide FF, which are neuropeptides,have recently been clarified and furthermore, low molecularphysiologically active lipids or nucleic acid derivatives such ascysteinyl leukotrienes, sphingosine-1-phosphate, lysophosphatidic acid,sphingosylphosphorylcholine, UDP-glucose, etc., have been identified tobe ligands to orphan receptors (Howard, A. D. et al., TiPS, 22, 132-140,2001). It was shown that MCH took part in obesity since its knockoutmice showed the reduced body weight and lean phenotype (Shimada, M. etal., Nature, 396, 670-674, 1998), and because its receptor was revealed,it became possible to explore a receptor antagonist likely to be anantiobesity agent. It is also reported that urotensin II shows a potentaction on the cardiocirculatory system, since it induces heart ischemiaby intravenous injection to monkey (Ames, R. S. et al., Nature, 401,282-286, 1999).

[0006] As described above, orphan receptors and ligands thereto oftentake part in a new physiological activity, and it is expected that theirclarification will lead to development of new drugs. However, it isknown that research on ligands to orphan receptors is accompanied bymany difficulties. For example, it is generally unknown what secondarysignal transduction system will take place after orphan receptorsexpressed on cells responded to ligands, and various response systemshould be examined. Moreover, tissues where ligands are present are notreadily predictable so that various tissue extracts should be prepared.Furthermore, since an amount of ligand required to stimulate itsreceptor is sufficient even in an extremely low concentration when theligand is a peptide, the amount of such a ligand present in vivo is atrace amount in many cases. In addition, a peptide is digested bypeptidase to lose its activity, or undergoes non-specific adsorption sothat its recovery becomes poor during purification. Normally, it is thusextremely difficult to extract such a ligand from the living body andisolate an amount of the ligand necessary for determination of itsstructure. The presence of many orphan receptors was unraveled, but onlya very small part of ligands to these receptors were discovered so fardue to the foregoing problems.

[0007] GPR7 is one of the reported orphan G protein-coupled receptors(SEQ ID NO:49, O'Dowd, B. F. et al., Genomics, 28, 84-91, 1995). GPR7has a low homology to somatostatin receptor (SSTR3) and opioid receptors(δ, κ and μ). Also, GPR7 is found to have a homology of about 64% toGPR8 (SEQ ID NO:66, O'Dowd, B. F. et al., Genomics, 28, 84-91, 1995) onan amino acid level. It is reported by O'Dowd, B. F. et al. that [³H]bremazocine binds to the membrane fraction of GPR7 and this binding isinhibited by β-funaltrexamine, [D-Pro4]morphiceptin or β-endorphin,which is a μ-opioid receptor selective ligand, U50 or 488, which is aκ-opioid receptor selective ligand, or naltrindole, which is a δ-opioidreceptor selective ligand.

[0008] The present invention provides a novel peptide capable of bindingto GPR7, etc., its DNA, a method of screening a drug using the peptideand GPR7, etc.

DISCLOSURE OF THE INVENTION

[0009] The present inventors made extensive studies to solve theforegoing problems. As a result, the inventors succeeded in acquiringDNAs encoding novel peptides (GPR7 ligands) capable of binding to GPR7,from human whole brain, mouse whole brain and rat whole brain, and foundthat the GPR7 ligands exhibit an appetite (eating) stimulating activity.In addition, the inventors succeeded in acquiring DNAs encoding GPR7 andGPR8, respectively, from bovine hypothalamus. As a result of furtherstudies based on these findings, the inventors have come to accomplishthe present invention.

[0010] That is, the present invention provides the following features:

[0011] (1) A peptide containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:1wherein the N-terminal amino acid residue may optionally be brominated,or its amide or ester, or a salt thereof;

[0012] (2) The peptide or its amide or ester, or a salt thereof,according to (1), which contains the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:1;

[0013] (3) The peptide or its amide or ester, or a salt thereof,according to (1), which has the amino acid sequence represented by SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:66;

[0014] (4) The peptide or its amide or ester, or a salt thereof,according to (1), wherein the N-terminal tryptophan residue is6-brominated and which has the amino acid sequence represented by SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:66;

[0015] (5) A peptide containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:4wherein the N-terminal amino acid residue may optionally be brominated,or its amide or ester, or a salt thereof;

[0016] (6) The peptide or its amide or ester, or a salt thereof,according to (5), which contains the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:4;

[0017] (7) The peptide or its amide or ester, or a salt thereof,according to (5), which has the amino acid sequence represented by SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:67;

[0018] (8) The peptide or its amide or ester, or a salt thereof,according to (5), wherein the N-terminal tryptophan residue is6-brominated and which has the amino acid sequence represented by SEQ IDNO:67;

[0019] (9) The peptide or its amide or ester, or a salt thereof,according to (5), wherein the N-terminal tryptophan residue is6-brominated and which has the amino acid sequence represented by SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:67;

[0020] (10) A peptide containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:7 wherein the N-terminal amino acid residue may optionally bebrominated, or its amide or ester, or a salt thereof;

[0021] (11) The peptide or its amide or ester, or a salt thereof,according to (10), which contains the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:7;

[0022] (12) The peptide or its amide or ester, or a salt thereof,according to (10), which has the amino acid sequence represented by SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:68 or SEQ ID NO:69;

[0023] (13) A peptide containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:13 wherein the N-terminal amino acid residue may optionally bebrominated, or its amide or ester, or a salt thereof;

[0024] (14) The peptide or its amide or ester, or a salt thereof,according to (13), which contains the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:13;

[0025] (15) The peptide or its amide or ester, or a salt thereof,according to (13), which has the amino acid sequence represented by SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:70;

[0026] (16) A peptide containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:16 wherein the N-terminal amino acid residue may optionally bebrominated, or its amide or ester, or a salt thereof;

[0027] (17) The peptide or its amide or ester, or a salt thereof,according to (16), which contains the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:16;

[0028] (18) The peptide or its amide or ester, or a salt thereof,according to (17), which has the amino acid sequence represented by SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:71;

[0029] (19) The peptide or its amide or ester, or a salt thereof,according to (1) through (18), which is capable of binding to a proteinor its salt containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:49 or SEQID NO:86;

[0030] (20) The peptide or its amide or ester, or a salt thereof,according to (1) through (18), which is capable of binding to a proteinor its salt containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:59;

[0031] (21) The peptide or its amide or ester, or a salt thereof,according to (1) through (18), which is capable of binding to a proteinor its salt containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:84 or SEQID NO:88;

[0032] (22) A partial peptide of the peptide according to any one of (1)through (21), or its amide or ester, or a salt thereof;

[0033] (23) A precursor peptide of the peptide according to any one of(1) through (21), or its amide or ester, or a salt thereof;

[0034] (24) The precursor peptide or its amide or ester, or a saltthereof, according to (23), which contains the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:19;

[0035] (25) The peptide or its amide or ester, or a salt thereof,according to (24), which has the amino acid sequence represented by SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:72;

[0036] (26) The peptide or its amide or ester, or a salt thereof,according to (23), which contains the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:22;

[0037] (27) The peptide or its amide or ester, or a salt thereof,according to (26), which has the amino acid sequence represented by SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:73;

[0038] (28) A polynucleotide containing a polynucleotide encoding thepeptide according to any one of (1) through (21);

[0039] (29) The polynucleotide according to (28), which has the basesequence represented by SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ IDNO:79;

[0040] (30) A polynucleotide containing a polynucleotide encoding thepartial peptide according to (22);

[0041] (31) A polynucleotide containing a polynucleotide encoding theprecursor peptide according to (23);

[0042] (32) The polynucleotide according to (31), which has the basesequence represented by SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:80 or SEQ ID NO:81;

[0043] (33) The polynucleotide according to (28) to (32), which is aDNA;

[0044] (34) A recombinant vector containing the polynucleotide accordingto any one of (28) to (33);

[0045] (35) A transformant transformed with the recombinant vectoraccording to (34);

[0046] (36) A method of manufacturing the peptide, its partial peptideor its precursor peptide, or a salt thereof, according to any one of (1)to (21), which comprises culturing the transformant according to (35)and producing the peptide, partial peptide or precursor peptideaccording to any one of (1) to (21);

[0047] (37) An antibody to the peptide, its partial peptide or itsprecursor peptide, or its amide or ester, or a salt thereof, accordingto any one of (1) to (21);

[0048] (38) The antibody according to (37), which is a neutralizingantibody to inactivate the activity of the peptide, its partial peptide,or its amide or ester, or a salt thereof, according to any one of (1) to(21);

[0049] (39) A pharmaceutical comprising the antibody according to (37);

[0050] (40) The pharmaceutical according to (39), which is apreventive/therapeutic agent for obesity or hyperphagia;

[0051] (41) A diagnostic product comprising the antibody according to(37);

[0052] (42) The diagnostic product according to (41), which is adiagnostic product for anorexia, obesity or hyperphagia;

[0053] (43) A pharmaceutical comprising the peptide, its partialpeptide, or its amide or ester, or a salt thereof, according to any oneof (1) to (21);

[0054] (44) The pharmaceutical according to (43), which is apreventive/therapeutic agent for anorexia or eating stimulant;

[0055] (45) A pharmaceutical comprising the polynucleotide according to(28);

[0056] (46) The pharmaceutical according to (45), which is apreventive/therapeutic agent for anorexia or eating stimulant;

[0057] (47) A diagnostic product comprising the polynucleotide accordingto (28);

[0058] (48) The diagnostic product according to (47), which is adiagnostic product for anorexia, obesity or hyperphagia;

[0059] (49) A polynucleotide containing a complementary base sequence tothe polynucleotide according to (28), or a part thereof;

[0060] (50) A pharmaceutical comprising the polynucleotide according to(49);

[0061] (51) The pharmaceutical according to (50), which is apreventive/therapeutic agent for obesity or hyperphagia;

[0062] (52) A method of screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:49,which comprises using the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:49;

[0063] (53) A method of screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:59,which comprises using the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:59;

[0064] (54) A method of screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:84,which comprises using the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:84;

[0065] (55) A kit for screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:49,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:49;

[0066] (56) A kit for screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:59,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:59;

[0067] (57) A kit for screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:84,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:84;

[0068] (58) A compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:49, which isobtainable by using the screening method according to (52) or thescreening kit according to (55);

[0069] (59) A compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:59, which isobtainable by using the screening method according to (53) or thescreening kit according to (56);

[0070] (60) A compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:84, which isobtainable by using the screening method according to (54) or thescreening kit according to (57);

[0071] (61) The compound or its salt according to (58) to (60), which isan agonist;

[0072] (62) The compound or its salt according to (58) to (60), which isan antagonist;

[0073] (63) A pharmaceutical comprising the compound or its saltaccording to any one of (58) to (60);

[0074] (64) A preventive/therapeutic agent for anorexia or eatingstimulant comprising the agonist according to (61);

[0075] (65) A preventive/therapeutic agent for obesity or hyperphagiacomprising; the antagonist according to (62);

[0076] (66) An antiobesity agent obtainable by using the screeningmethod according to (52) or the screening kit according to (55);

[0077] (67) An antiobesity agent obtainable by using the screeningmethod according to (53) or the screening kit according to (56);

[0078] (68) An antiobesity agent obtainable by using the screeningmethod according to (54) or the screening kit according to (57);

[0079] (69) A method of screening a compound or its salt that alters theexpression level of the peptide, its partial peptide or its precursorpeptide according to any one of (1) to (21), which comprises using a DNAencoding the peptide, its partial peptide or its precursor peptideaccording to any one of (1) to (21);

[0080] (70) A kit for screening a compound or its salt that alters theexpression level of the peptide, its partial peptide or its precursorpeptide according to any one of (1) to (21), comprising a DNA encodingthe peptide, its partial peptide or its precursor peptide according toany one of (1) to (21);

[0081] (71) A compound or its salt that alters the expression level ofthe peptide, its partial peptide or its precursor peptide according toany one of (1) to (21), which is obtainable by using the screeningmethod according to (69) or the screening kit according to (70);

[0082] (72) The compound or its salt according to (71), which is acompound or its salt that increases the expression level;

[0083] (73) The compound or its salt according to (71), which is acompound or its salt that decreases the expression level;

[0084] (74) A pharmaceutical comprising the compound or its saltaccording to (71);

[0085] (75) A preventive/therapeutic agent for anorexia or eatingstimulant comprising the compound or its salt according to (72);

[0086] (76) A preventive/therapeutic agent for obesity or hyperphagiacomprising the compound or its salt according to (73);

[0087] (77) A method for preventing/treating anorexia, which comprisesadministering to a mammal an effective amount of the peptide, itspartial peptide, or its amide or ester, or a salt thereof, according toany one of (1) to (21), the polynucleotide according to (28), theagonist according to (61), or the compound or its salt according to(72);

[0088] (78) A method for stimulating appetite, which comprisesadministering to a mammal an effective amount of the peptide, itspartial peptide, or its amide or ester, or a salt thereof, according toany one of (1) to (21), the polynucleotide according to (28), theagonist according to (61), or the compound or its salt according to(72);

[0089] (79) A method for preventing/treating obesity or hyperphagia,which comprises administering to a mammal an effective amount of theantibody according to (37), the polynucleotide according to (49), theantagonist according to (62), or the compound or its salt according to(73);

[0090] (80) A protein or its salt containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:86;

[0091] (81) The protein or its salt according to (80), containing theamino acid sequence represented by SEQ ID NO:86;

[0092] (82) A partial peptide or its salt of the protein according to(80);

[0093] (83) A polynucleotide containing a polynucleotide encoding theprotein according to (80), or a partial peptide thereof;

[0094] (84) The polynucleotide according to (83), which is a DNA;

[0095] (85) The polynucleotide according to (84), which contains thebase sequence represented by SEQ ID NO:87;

[0096] (86) A recombinant vector containing the polynucleotide accordingto (83);

[0097] (87) A transformant transformed by the recombinant vectoraccording to (86);

[0098] (88) A method of manufacturing the protein according to (80), itspartial peptide or a salt thereof, which comprises culturing thetransformant according to (87) and producing the protein according to(80), its partial peptide or a salt thereof;

[0099] (89) A pharmaceutical comprising the protein according to (80) orthe partial peptide according to (82), or a salt thereof;

[0100] (90) A pharmaceutical comprising the polynucleotide according to(83);

[0101] (91) The pharmaceutical according to (90), which is apreventive/therapeutic agent for anorexia or eating stimulant;

[0102] (92) A diagnostic product comprising the polynucleotide accordingto (83);

[0103] (93) The diagnostic product according to (92), which is adiagnostic product for anorexia, obesity or hyperphagia;

[0104] (94) An antibody to the protein according to (80) or the partialpeptide according to (82), or a salt thereof;

[0105] (95) The antibody according to (94), which is a neutralizingantibody to inactivate signal transduction of the protein according to(80);

[0106] (96) A pharmaceutical comprising the antibody according to (94);

[0107] (97) The pharmaceutical according to (96), which is apreventive/therapeutic agent for obesity or hyperphagia;

[0108] (98) A diagnostic product comprising the antibody according to(94);

[0109] (99) The diagnostic product according to (99), which is adiagnostic product for anorexia, obesity or hyperphagia;

[0110] (100) A polynucleotide containing a complementary base sequenceto the polynucleotide according to (83), or a part thereof;

[0111] (101) A pharmaceutical comprising the polynucleotide according to(100);

[0112] (102) The pharmaceutical according to (101), which is apreventive/therapeutic agent for obesity or hyperphagia;

[0113] (103) A protein or its salt containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:88;

[0114] (104) The protein or its salt according to (103), which containsthe amino acid sequence represented by SEQ ID NO:88;

[0115] (105) A partial peptide of the protein according to (103), or asalt thereof;

[0116] (106) A polynucleotide containing a polynucleotide encoding theprotein according to (103) or a partial peptide thereof;

[0117] (107) The polynucleotide according to (106), which is a DNA;

[0118] (108) The polynucleotide according to (107), which contains thebase sequence represented by SEQ ID NO:89;

[0119] (109) A recombinant vector containing the polynucleotideaccording to (108);

[0120] (110) A transformant transformed with the recombinant vectoraccording to (109);

[0121] (111) A method of manufacturing the protein according to (103),its partial peptide, or a salt thereof, which comprises culturing thetransformant of (110) and producing the protein according to (103) orits partial peptide;

[0122] (112) A pharmaceutical comprising the protein according to (103)or the partial peptide according to (105), or a salt thereof;

[0123] (113) A pharmaceutical comprising the polynucleotide according to(106);

[0124] (114) The pharmaceutical according to (113), which is apreventive/therapeutic agent for anorexia or eating stimulant;

[0125] (115) A diagnostic product comprising the polynucleotideaccording to (106);

[0126] (116) The diagnostic product according to (115), which is adiagnostic product for anorexia, obesity or hyperphagia;

[0127] (117) An antibody to the protein according to (103) or thepartial peptide according to (105), or a salt thereof;

[0128] (118) The antibody according to (117), which is a neutralizingantibody to inactivate signal transduction of the protein according to(103);

[0129] (119) A pharmaceutical comprising the antibody according to(117);

[0130] (120) The pharmaceutical according to (119), which is apreventive/therapeutic agent for obesity or hyperphagia;

[0131] (121) A diagnostic product comprising the antibody according to(117);

[0132] (122) The diagnostic product according to (121), which is adiagnostic product for anorexia, obesity or hyperphagia;

[0133] (123) A polynucleotide containing a complementary base sequenceto the polynucleotide according to (106), or a part thereof;

[0134] (124) A pharmaceutical comprising the polynucleotide according to(123);

[0135] (125) The pharmaceutical according to (124), which is apreventive/therapeutic agent for obesity or hyperphagia;

[0136] (126) A method of screening a compound or its salt that altersthe binding property between the peptide, its partial peptide, or itsamide or ester, or a salt thereof, according to any one of (1) to (21)and a protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:86, which comprises using the peptide, its partial peptide, or itsamide or ester, or a salt thereof, according to any one of (1) to (21)and a protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:86;

[0137] (127) A method of screening a compound or its salt that altersthe binding property between the peptide, its partial peptide, or itsamide or ester, or a salt thereof, according to any one of (1) to (21)and a protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:88, which comprises using the peptide, its partial peptide, or itsamide or ester, or a salt thereof, according to any one of (1) to (21)and a protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:88;

[0138] (128) A kit for screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:86,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:86;

[0139] (129) A kit for screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:88,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:88;

[0140] (130) A compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:86, which isobtainable by using the screening method according to (126) or thescreening kit according to (128);

[0141] (131) A compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:88, which isobtainable by using the screening method according to (127) or thescreening kit according to (129);

[0142] (132) The compound or its salt according to (130) or (131), whichis an agonist;

[0143] (133) The compound or its salt according to (130) or (131), whichis an antagonist;

[0144] (134) A pharmaceutical comprising the compound or its saltaccording to (130) or (131);

[0145] (135) A preventive/therapeutic agent for anorexia or eatingstimulant, comprising the agonist according to (132);

[0146] (136) A preventive/therapeutic agent for obesity or hyperphagia,comprising the antagonist according to (133);

[0147] (137) A method of screening a compound or its salt that altersthe expression level of a protein containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:86, which comprises using a DNA encoding a protein containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:86;

[0148] (138) A kit for screening a compound or its salt that alters theexpression level of a protein containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:86, comprising a DNA encoding a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:86;

[0149] (139) A compound or its salt that alters the expression level ofa protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:86, whichis obtainable by using the screening method according to (137) or thescreening kit according to (138);

[0150] (140) The compound or its salt according to (139), which is acompound or its salt that increases the expression level;

[0151] (141) The compound or its salt according to (139), which is acompound or its salt that decreases the expression level;

[0152] (142) A pharmaceutical comprising the compound or its saltaccording to (139);

[0153] (143) A preventive/therapeutic agent for anorexia or eatingstimulant comprising the compound or its salt according to (140);

[0154] (144) A preventive/therapeutic agent for obesity or hyperphagiacomprising the compound or its salt according to (141);

[0155] (145) A method of screening a compound or its salt that altersthe expression level of a protein containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:88, which comprises using a DNA encoding a protein containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:88;

[0156] (146) A kit for screening a compound or its salt that alters theexpression level of a protein containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:88, comprising a DNA encoding a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:88;

[0157] (147) A compound or its salt that alters the expression level ofa protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:88, whichis obtainable by using the screening method according to (145) or thescreening kit according to (146);

[0158] (148) The compound or its salt according to (147), which is acompound or its salt that increases the expression level;

[0159] (149) The compound or its salt according to (147), which is acompound or its salt that decreases the expression level;

[0160] (150) A pharmaceutical comprising the compound or its saltaccording to (147);

[0161] (151) A preventive/therapeutic agent for anorexia or eatingstimulant comprising the compound or its salt according to (148);

[0162] (152) A preventive/therapeutic agent for obesity or hyperphagiacomprising the compound or its salt according to (149);

[0163] (153) A method for preventing/treating anorexia, which comprisesadministering to a mammal an effective amount of the protein accordingto (80), its partial peptide or a salt thereof, the polynucleotideaccording to (83), the protein according to (103), its partial peptideor a salt thereof, the polynucleotide according to (106), the agonistaccording to (132), the compound or its salt according to (140), or thecompound or its salt according to (148);

[0164] (154) A method for stimulating appetite, which comprisesadministering to a mammal an effective amount of the protein accordingto (80), its partial peptide or a salt thereof, the polynucleotideaccording to (83), the protein according to (103), its partial peptideor a salt thereof, the polynucleotide according to (106), the agonistaccording to (132), the compound or its salt according to (140), or thecompound or its salt according to (148);

[0165] (155) A method for preventing/treating obesity or hyperphagia,which comprises administering to a mammal an effective amount of theantibody according to (94), the polynucleotide according to (100), theantibody according to (117), the polynucleotide according to (123), theantagonist according to (133), the compound or its salt according to(141), or the compound or its salt according to (149);

[0166] (156) A non-human mammal bearing the DNA according to (28), whichis exogenous, or its variant DNA;

[0167] (157) The mammal according to (156), wherein the non-human mammalis a rodent;

[0168] (158) A recombinant vector bearing the exogenous DNA or itsvariant DNA according to (28) and capable of expressing in a mammal;

[0169] (159) A non-human embryonic stem cell, wherein the DNA accordingto (28) is inactivated;

[0170] (160) The embryonic stem cell according to (159), wherein the DNAis inactivated by introducing a reporter gene;

[0171] (161) The embryonic stem cell according to (159), wherein thenon-human mammal is a rodent;

[0172] (162) A non-human mammal deficient in expressing the DNAaccording to (28), wherein the DNA is inactivated;

[0173] (163) A non-human mammal according to (162), wherein the DNA isinactivated by inserting a reporter gene therein and the reporter geneis capable of expressing under control of a promoter for the DNAaccording to (28);

[0174] (164) The non-human mammal according to (162), which is a rodent;

[0175] (165) A method of screening a compound or its salt that promotesor inhibits the activity of a promoter for the DNA according to (28),which comprises administering a test compound to the mammal according to(163) and detecting expression of the reporter gene;

[0176] (166) A non-human mammal bearing the DNA according to (83), whichis exogenous, or its variant DNA;

[0177] (167) The mammal according to (166), wherein the non-human mammalis a rodent;

[0178] (168) A recombinant vector bearing the exogenous DNA or itsvariant DNA according to (83) and capable of expressing in a mammal;

[0179] (169) A non-human embryonic stem cell, wherein the DNA accordingto (83) is inactivated;

[0180] (170) The embryonic stem cell according to (169), wherein the DNAis inactivated by introducing a reporter gene;

[0181] (171) The embryonic stem cell according to (169), wherein thenon-human mammal is a rodent;

[0182] (172) A non-human mammal deficient in expressing the DNAaccording to (83), wherein the DNA is inactivated;

[0183] (173) A non-human mammal according to (172), wherein the DNA isinactivated by inserting a reporter gene therein and the reporter geneis capable of expressing under control of a promoter for the DNAaccording to (83);

[0184] (174) The non-human mammal according to (172), which is a rodent;

[0185] (175) A method of screening a compound or its salt that promotesor inhibits the activity of a promoter for the DNA according to (83),which comprises administering a test compound to the mammal according to(173) and detecting expression of the reporter gene;

[0186] (176) A non-human mammal bearing the DNA according to (106),which is exogenous, or its variant DNA;

[0187] (177) The mammal according to (176), wherein the non-human mammalis a rodent;

[0188] (178) A recombinant vector bearing the exogenous DNA according to(106) or its variant DNA and capable of expressing in a mammal;

[0189] (179) A non-human embryonic stem cell, wherein the DNA accordingto (106) is inactivated;

[0190] (180) The embryonic stem cell according to (179), wherein the DNAis inactivated by introducing a reporter gene;

[0191] (181) The embryonic stem cell according to (179), wherein thenon-human mammal is a rodent;

[0192] (182) A non-human mammal deficient in expressing the DNAaccording to (106), wherein the DNA is inactivated;

[0193] (183) The non-human mammal according to (182), wherein the DNA isinactivated by inserting a reporter gene therein and the reporter geneis capable of expressing under control of a promoter for the DNAaccording to (106);

[0194] (184) The non-human mammal according to (182), which is a rodent;

[0195] (185) A method of screening a compound or its salt that promotesor inhibits the activity of a promoter for the DNA according to (106),which comprises administering a test compound to the mammal according to(183) and detecting expression of the reporter gene;

[0196] (186) Use of the peptide, its partial peptide, its amide orester, or a salt thereof, according to any one of (1) through (21), thepolynucleotide according to (28), the agonist according to (61), or thecompound or its salt according to (72), for manufacturing apreventive/therapeutic agent for anorexia;

[0197] (187) Use of the peptide, its partial peptide, its amide orester, or a salt thereof, according to any one of (1) through (21), thepolynucleotide according to (28), the agonist according to (61), or thecompound or its salt according to (72), for manufacturing an eatingstimulant;

[0198] (188) Use of the antibody according to (37), the polynucleotideaccording to (49), the antagonist according to (62), or the compound orits salt according to (73), for manufacturing a preventive/therapeuticagent for obesity or hyperphagia;

[0199] (189) Use of the protein according to (80) or its partial peptideor a salt thereof, the polynucleotide according to (83), the proteinaccording to (103) or its partial peptide or a salt thereof, thepolynucleotide according to (106), the agonist according to (132), thecompound or its salt according to (140) or the compound or its saltaccording to (148), for manufacturing a preventive/therapeutic agent foranorexia;

[0200] (190) Use of the protein according to (80) or its partial peptideor a salt thereof, the polynucleotide according to (83), the proteinaccording to (103) or its partial peptide or a salt thereof, thepolynucleotide according to (106), the agonist according to (132), thecompound or its salt according to (140) or the compound or its saltaccording to (148), for manufacturing an eating stimulant; and,

[0201] (191) Use of the antibody according to (94), the polynucleotideaccording to (100), the antibody according to (117), the polynucleotideaccording to (123), the antagonist according to (133), the compound orits salt according to (141), or the compound or its salt according to(149), for manufacturing a preventive/therapeutic agent for obesity orhyperphagia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0202]FIG. 1 shows the DNA sequence of human GPR7 ligand precursor H.

[0203]FIG. 2 shows the amino acid sequence of human GPR7 ligandprecursor H.

[0204]FIG. 3 shows the DNA sequence of mouse GPR7 ligand precursor H.

[0205]FIG. 4 shows the amino acid sequence of mouse GPR7 ligandprecursor H.

[0206]FIG. 5 shows the DNA sequence of rat GPR7 ligand precursor H.

[0207]FIG. 6 shows the amino acid sequence of rat GPR7 ligand precursorH.

[0208]FIG. 7 shows a comparison between human, rat and mouse GPR7 ligandprecursors H, wherein the matched amino acids are indicted in a box, andthe arrow indicates the predicted cleavage site of a secretion signal.

[0209]FIG. 8 shows the results of suppression detected on a luciferaseactivity by ligand stimulation, when the culture supernatants of ligandexpression vector pAK-S64 and empty expression vector(pAKKO-111H)-expressed CHO cells were added in the presence of forskolin(FSK) to a medium of CHO cells wherein GPR7 cDNA-inserted plasmid wastransiently expressed.

[0210]FIG. 9 shows the results of suppression detected on a luciferaseactivity, when he culture supernatant of CHO cells wherein S64 wastransiently expressed was added in the presence of forskolin (FSK) to amedium of CHO cells wherein TGR26 was transiently expressed.

[0211]FIG. 10 shows the results of GPR7-expressed CHO cell-specific cAMPproduction level suppression caused by the supernatant of cells whereinS64 was transiently expressed.

[0212]FIG. 11 shows the results of cAMP production level suppression ofmock CHO caused by the supernatant of cells wherein S64 was transientlyexpressed.

[0213]FIG. 12 shows the tissue distribution of GPR7 ligand mRNA in ratand the results of its expression level as determined by RT-PCR.

[0214]FIG. 13 shows a chromatogram of endogenous GPR7 ligand finallypurified from bovine hypothalamus, which indicates the chromatographicpattern on μRPC C2/C18 SC 2.1/10 at the final step of purification andthe cAMP production level assayed using the cAMP-screen system (ABI)after reacting each fraction with human GPR7-expressed CHO cells. Thechromatographic pattern shows the absorbance at 215 nm and the elutionconcentration of acetonitrile.

[0215]FIG. 14 shows the results of N-terminal sequencing of theendogenous GPR7 ligand purified from bovine hypothalamus.

[0216]FIG. 15 shows an ESIMS spectrum (upper column) and a MS/MSspectrum (lower column) of the endogenous GPR7 ligand purified frombovine hypothalamus.

[0217]FIG. 16 shows a zoom scanning spectrum of trivalent molecularions.

[0218]FIG. 17 shows the results of standard analysis performed by mixingPTH-5-bromotryptophan (5BrW) and PTH-6-bromotryptophan (6BrW) with 20amino acid PTH standards (peaks shown by asterisk*), whereby it wasconfirmed that the peaks of standard 5BrW overlapped with those of 5BrWin an unknown sample.

[0219]FIG. 18 shows the results of standard analysis performed by mixingPTH-5-bromotryptophan (5BrW) and PTH-6-bromotryptophan (6BrW) with 20amino acid PTH standards (peaks shown by asterisk*), whereby it wasconfirmed that the peaks of standard 6BrW overlapped with those of 6BrWin an unknown sample.

[0220]FIG. 19 shows the results of N-terminal sequencing of GPR7Lpurified from bovine hypothalamus, wherein the amino acids by standardanalysis and at cycle 1 on the chromatogram up to cycle 2 coincided withthe peaks of 6-bromotryptophan.

[0221]FIG. 20 shows a DNA sequence of bovine GPR7 ligand precursor H.

[0222]FIG. 21 shows an amino acid sequence of bovine GPR7 ligandprecursor H.

[0223]FIG. 22 shows change in feed uptake with passage of time every 2hours after non-brominated GPR7L or distilled water was administered torat into the lateral ventricle, wherein Vehicle and bGPR7L (Br—)indicate distilled water and non-brominated bovine GPR7 ligand,respectively.

[0224]FIG. 23 shows a FMS spectrum of endogenous GPR7 ligand.

[0225]FIG. 24 shows the results of the tissue distribution andexpression level of GPR7 ligand mRNA in human as determined by RT-PCR.

[0226]FIG. 25 shows the results of the tissue distribution andexpression level of rat GPR7 (rat TGR26) mRNA as determined by RT-PCR.

[0227]FIG. 26 shows a cDNA sequence of bovine GPR7.

[0228]FIG. 27 shows an amino acid sequence of bovine GPR7.

[0229]FIG. 28 shows a cDNA sequence of bovine GPR8.

[0230]FIG. 29 shows an amino acid sequence of bovine GPR8.

[0231]FIG. 30 shows the results of final purification of human GPR7ligand from the culture supernatant of human GPR7 ligand-expressed CHOcells, wherein a chromatographic pattern of μRPC C2/C18 SC2.1/10 at thefinal purification step and the specific intracellular cAMP productionsuppression activity obtained by reacting each fraction with the humanGPR7-expressed CHO cells are shown. On the chromatogram, absorbance at215 nm and the elution concentration of acetonitrile are shown.

[0232]FIG. 31 shows the results of N-terminal sequencing of GPR7 ligandpurified from the culture supernatant of human GPR7 ligand-expressed CHOcells.

[0233]FIG. 32 shows an ESI-MS spectrum of GPR7 ligand purified from theculture supernatant of human GPR7 ligand-expressed CHO cells.

[0234]FIG. 33 shows the results of Scatchard analysis using human GPR7ligand-expressed CHO cells.

BEST MODE FOR CARRYING OUT THE INVENTION

[0235] The peptide of the present invention having the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13 orSEQ ID NO:16 (hereinafter sometimes merely referred to as the peptide ofthe present invention) may be any peptide derived from any cells ofhuman or other warm-blooded animals, e.g., guinea pigs, rats, mice,chicken, rabbits, swine, sheep, bovine, monkeys, etc. (e.g., retinacells, liver cells, splenocytes, nerve cells, glial cells, β cells ofpancreas, bone marrow cells, mesangial cells, Langerhans' cells,epidermic cells, epithelial cells, endothelial cells, fibroblasts,fibrocytes, myocytes, fat cells, immune cells (e.g., macrophages, Tcells, B cells, natural killer cells, mast cells, neutrophils,basophils, eosinophils, monocytes), megakaryocyte, synovial cells,chondrocytes, bone cells, osteoblasts, osteoclasts, mammary gland cells,hepatocytes or interstitial cells, the corresponding precursor cells,stem cells, cancer cells, etc., of these cells), or any tissues wheresuch cells are present, e.g., brain or any region of the brain (e.g.,retina, olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus,thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum),spinal cord, hypophysis, stomach, pancreas, kidney, liver, gonad,thyroid, gall-bladder, bone marrow, adrenal gland, skin, muscle, lung,gastrointestinal tract (e.g., large intestine and small intestine),blood vessel, heart, thymus, spleen, submandibular gland, peripheralblood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletalmuscle, etc.; or hemocyte type cells or their cultured cells (e.g., MEL,M1, CTLL-2, HT-2, WEHI-3, HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4,MOLT-10, CCRF-CEM, TALL-1, Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78,HUT-102, H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG-01, etc.). Thepeptide may also be a synthetic peptide.

[0236] The amino acid sequence which has substantially the same aminoacid sequence as that represented by SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:13 or SEQ ID NO:16 includes an amino acid sequencehaving at least about 70% homology, preferably at least about 80%homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to the amino acid sequencerepresented by SEQ ID NO:1.

[0237] As the peptide having substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:1, preferred is apeptide having substantially the same amino acid sequence as the aminoacid sequence represented by SEQ ID NO:1 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:1, etc.

[0238] As the peptide having substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:4, preferred is apeptide having substantially the same amino acid sequence as the aminoacid sequence represented by SEQ ID NO:4 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:4, etc.

[0239] As the peptide having substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:7, preferred is apeptide having substantially the same amino acid sequence as the aminoacid sequence represented by SEQ ID NO:7 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:7, etc.

[0240] As the peptide having substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:13, preferred is apeptide having substantially the same amino acid sequence as the aminoacid sequence represented by SEQ ID NO:13 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:13, etc.

[0241] As the peptide having substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:16, preferred is apeptide having substantially the same amino acid sequence as the aminoacid sequence represented by SEQ ID NO:16 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:16, etc.

[0242] Specifically, the substantially equivalent activity includesactivities that the peptide of the present invention possesses (forexample, preventive/therapeutic activities for diseases described below,GPR7 binding activities, cell stimulating activities on theGPR7-expressed cells (e.g., activities that promote arachidonic acidrelease, acetylcholine release, intracellular Ca²⁺ release,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, change in cell membrane potential, phosphorylationof intracellular proteins, activation of c-fos, pH reduction, GTPγSbinding activities, etc.) and the like.

[0243] The term substantially equivalent is used to mean that theseactivities are equivalent in nature (e.g., biochemically orpharmacologically).

[0244] Specific examples of the amino acid sequence, which issubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:1, include:

[0245] (i) the amino acid sequence represented by SEQ ID NO:1;

[0246] (ii) the amino acid sequence represented by SEQ ID NO:2;

[0247] (iii) the amino acid sequence represented by SEQ ID NO:3;

[0248] (iv) the amino acid sequence represented by SEQ ID NO:66;

[0249] (v) the amino acid sequence represented by SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:66, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are deleted;

[0250] (vi) the amino acid sequence represented by SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:66, to which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are added;

[0251] (vii) the amino acid sequence represented by SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:66, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are inserted;

[0252] (viii) the amino acid sequence represented by SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:66, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are replacedby other amino acids;

[0253] (ix) amino acid sequences in combination of (v) to (viii) above;etc.

[0254] Specific examples of the amino acid sequence, which issubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:4, include:

[0255] (i) the amino acid sequence represented by SEQ ID NO:4;

[0256] (ii) the amino acid sequence represented by SEQ ID NO:5;

[0257] (iii) the amino acid sequence represented by SEQ ID NO:6;

[0258] (iv) the amino acid sequence represented by SEQ ID NO:67;

[0259] (v) the amino acid sequence represented by SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 or SEQ ID NO:67, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are deleted;

[0260] (vi) the amino acid sequence represented by SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 or SEQ ID NO:67, to which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are added;

[0261] (vii) the amino acid sequence represented by SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 or SEQ ID NO:67, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are inserted;

[0262] (viii) the amino acid sequence represented by SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 or SEQ ID NO:67, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are replacedby other amino acids;

[0263] (ix) amino acid sequences in combination of (v) to (viii) above;etc.

[0264] Specific examples of the amino acid sequence, which issubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:7, include:

[0265] (i) the amino acid sequence represented by SEQ ID NO:7;

[0266] (ii) the amino acid sequence represented by SEQ ID NO:8;

[0267] (iii) the amino acid sequence represented by SEQ ID NO:9;

[0268] (iv) the amino acid sequence represented by SEQ ID NO:10;

[0269] (v) the amino acid sequence represented by SEQ ID NO:11;

[0270] (vi) the amino acid sequence represented by SEQ ID NO:12;

[0271] (vii) the amino acid sequence represented by SEQ ID NO:68;

[0272] (viii) the amino acid sequence represented by SEQ ID NO:69;

[0273] (ix) the amino acid sequence represented by SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:68 or SEQ ID NO:69, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are deleted;

[0274] (x) the amino acid sequence represented by SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:68 or SEQ ID NO:69, to which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are added;

[0275] (xi) the amino acid sequence represented by SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:68 or SEQ ID NO:69, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are inserted;

[0276] (xii) the amino acid sequence represented by SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:68 or SEQ ID NO:69, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are replaced byother amino acids;

[0277] (xiii) amino acid sequences in combination of (ix) to (xii)above; etc.

[0278] Specific examples of the amino acid sequence, which issubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:13, include:

[0279] (i) the amino acid sequence represented by SEQ ID NO:13;

[0280] (ii) the amino acid sequence represented by SEQ ID NO:14;

[0281] (iii) the amino acid sequence represented by SEQ ID NO:15;

[0282] (iv) the amino acid sequence represented by SEQ ID NO:70;

[0283] (v) the amino acid sequence represented by SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 or SEQ ID NO:70, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are deleted;

[0284] (vi) the amino acid sequence represented by SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 or SEQ ID NO:70, to which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are added;

[0285] (vii) the amino acid sequence represented by SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 or SEQ ID NO:70, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 0.1) amino acids areinserted;

[0286] (viii) the amino acid sequence represented by SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15 or SEQ ID NO:70, in which 1 to 5 (preferably 1 to3, more preferably 1 to 2, and most preferably 1) amino acids arereplaced by other amino acids;

[0287] (ix) amino acid sequences in combination of (v) to (viii) above;etc.

[0288] Specific examples of the amino acid sequence, which issubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:16, include:

[0289] (i) the amino acid sequence represented by SEQ ID NO:16;

[0290] (ii) the amino acid sequence represented by SEQ ID NO:17;

[0291] (iii) the amino acid sequence represented by SEQ ID NO:18;

[0292] (iv) the amino acid sequence represented by SEQ ID NO:71;

[0293] (v) the amino acid sequence represented by SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18 or SEQ ID NO:71, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are deleted;

[0294] (vi) the amino acid sequence represented by SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18 or SEQ ID NO:71, to which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are added;

[0295] (vii) the amino acid sequence represented by SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18 or SEQ ID NO:71, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are inserted;

[0296] (viii) the amino acid sequence represented by SEQ ID NO:16, SEQID NO:17, SEQ ID NO:18 or SEQ ID NO:71, in which 1 to 5 (preferably 1 to3, more preferably 1 to 2, and most preferably 1) amino acids arereplaced by other amino acids;

[0297] (ix) amino acid sequences in combination of (v) to (viii) above;etc.

[0298] Specific examples of the peptide of the present invention are,for example:

[0299] [Peptide A]

[0300] human peptide having the amino acid sequence represented by SEQID NO:1;

[0301] mouse peptide having the amino acid sequence represented by. SEQID NO:2;

[0302] rat peptide having the amino acid sequence represented by SEQ IDNO:3;

[0303] bovine peptide having the amino acid sequence represented by SEQID NO:66;

[0304] [Peptide B]

[0305] human peptide having the amino acid sequence represented by SEQID NO:4;

[0306] mouse peptide having the amino acid sequence represented by SEQID NO:5;

[0307] rat peptide having the amino acid sequence represented by SEQ IDNO:6;

[0308] bovine peptide having the amino acid sequence represented by SEQID NO:67;

[0309] [Peptide C]

[0310] human peptide having the amino acid sequence represented by SEQID NO:7, or its amide;

[0311] mouse peptide having the amino acid sequence represented by SEQID NO:9, or its amide;

[0312] rat peptide having the amino acid sequence represented by SEQ IDNO:11, or its amide;

[0313] bovine peptide having the amino acid sequence represented by SEQID NO:68, or its amide;

[0314] [Peptide D]

[0315] human peptide having the amino acid sequence represented by SEQID NO:8;

[0316] mouse peptide having the amino acid sequence represented by SEQID NO:10;

[0317] rat peptide having the amino acid sequence represented by SEQ IDNO:12;

[0318] bovine peptide having the amino acid sequence represented by SEQID NO:69;

[0319] [Peptide E]

[0320] human peptide having the amino acid sequence represented by SEQID NO:13;

[0321] mouse peptide having the amino acid sequence represented by SEQID NO:14;

[0322] rat peptide having the amino acid sequence represented by SEQ IDNO:15;

[0323] bovine peptide having the amino acid sequence represented by SEQID NO:70;

[0324] [Peptide F]

[0325] human peptide having the amino acid sequence represented by SEQID NO:16;

[0326] mouse peptide having the amino acid sequence represented by SEQID NO:17;

[0327] rat peptide having the amino acid sequence represented by SEQ IDNO:18;

[0328] bovine peptide having the amino acid sequence represented by SEQID NO:71; and the like.

[0329] The partial peptide of the present invention may be any peptideso long as it is a partial peptide of the peptide of the presentinvention described above. Normally, peptides composed of at least 5amino acids, preferably at least 10 amino acids are preferred and thosefurther having activities similar to those of the peptide of the presentinvention are preferred.

[0330] The precursor peptide to the peptide of the present invention maybe a polypeptide including the peptide of the present inventiondescribed above, which can produce the peptide of the present inventionby cleaving with an appropriate peptidase.

[0331] Specifically, proteins, etc. having same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:19 or SEQ ID NO:22 are employed.

[0332] The protein having the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:19 or SEQID NO:22 may be any protein derived from any cells of human or otherwarm-blooded animals, e.g., guinea pigs, rats, mice, chicken, rabbits,swine, sheep, bovine, monkeys, etc. (e.g., retina cells, liver cells,splenocytes, nerve cells, glial cells, cells of pancreas, bone marrowcells, mesangial cells, Langerhans' cells, epidermic cells, epithelialcells, endothelial cells, fibroblasts, fibrocytes, myocytes, fat cells,immune cells (e.g., macrophages, T cells, B cells, natural killer cells,mast cells, neutrophils, basophils, eosinophils, monocytes),megakaryocyte, synovial cells, chondrocytes, bone cells, osteoblasts,osteoclasts, mammary gland cells, hepatocytes or interstitial cells, thecorresponding precursor cells, stem cells, cancer cells, etc., of thesecells), or any tissues where such cells are present, e.g., brain or anyregion of the brain (e.g., retina, olfactory bulb, amygdaloid nucleus,basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex,medulla oblongata, cerebellum), spinal cord, hypophysis, stomach,pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow,adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g., largeintestine and small intestine), blood vessel, heart, thymus, spleen,submandibular gland, peripheral blood, prostate, testis, ovary,placenta, uterus, bone, joint, skeletal muscle, etc.; or hemocyte typecells or their cultured cells (e.g., MEL, M1, CTLL-2, HT-2, WEHI-3,HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1,Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL,JK-1, CMK, KO-812, MEG-01, etc.). The protein may also be a syntheticprotein.

[0333] The amino acid sequence which has substantially the same aminoacid sequence as that represented by SEQ ID NO:19 or SEQ ID NO:22includes an amino acid sequence having at least about 70% homology,preferably at least about 80% homology, and more preferably at leastabout 90% homology, to the amino acid sequence represented by SEQ IDNO:19 or SEQ ID NO:22.

[0334] In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:19, include:

[0335] (i) the amino acid sequence represented by SEQ ID NO:19 (humantype);

[0336] (ii) the amino acid sequence represented by SEQ ID NO:20 (mousetype);

[0337] (iii) the amino acid sequence represented by SEQ ID NO:21 (rattype);

[0338] (iv) the amino acid sequence represented by SEQ ID NO:72 (bovinetype);

[0339] (v) the amino acid sequence represented by SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21 or SEQ ID NO:72, in which 1 to 15 (preferably 1 to10, more preferably 1 to 5, and most preferably 1 to 3) amino acids aredeleted;

[0340] (vi) the amino acid sequence represented by SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21 or SEQ ID NO:72, to which 1 to 15 (preferably 1 to10, more preferably 1 to 5, and most preferably 1 to 3) amino acids areadded;

[0341] (vii) the amino acid sequence represented by SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21 or SEQ ID NO:72, in which 1 to 15 (preferably 1 to10, more preferably 1 to 5, and most preferably 1 to 3) amino acids areinserted;

[0342] (viii) the amino acid sequence represented by SEQ ID NO:19, SEQID NO:20, SEQ ID NO:21 or SEQ ID NO:72, in which 1 to 15 (preferably 1to 10, more preferably 1 to 5, and most preferably 1 to 3) amino acidsare replaced by other amino acids;

[0343] (ix) amino acid sequences in combination of (v) to (viii) above;etc.

[0344] Examples of the amino acid sequence, which has substantially thesame amino acid sequence as that represented by SEQ ID NO:22, include:

[0345] (i) the amino acid sequence represented by SEQ ID NO:22 (humantype);

[0346] (ii) the amino acid sequence represented by SEQ ID NO:23 (mousetype);

[0347] (iii) the amino acid sequence represented by SEQ ID NO:24 (rattype);

[0348] (iv) the amino acid sequence represented by SEQ ID NO:73 (bovinetype);

[0349] (v) the amino acid sequence represented by SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24 or SEQ ID NO:73, in which 1 to 15 (preferably 1 to10, more preferably 1 to 5, and most preferably 1 to 3) amino acids aredeleted;

[0350] (vi) the amino acid sequence represented by SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24 or SEQ ID NO:73, to which 1 to 15 (preferably 1 to10, more preferably 1 to 5, and most preferably 1 to 3) amino acids areadded;

[0351] (vii) the amino acid sequence represented by SEQ ID NO:22, SEQ IDNO:23 or SEQ ID NO:24, in which 1 to 15 (preferably 1 to 10, morepreferably 1 to 5, and most preferably 1 to 3) amino acids are inserted;

[0352] (viii) the amino acid sequence represented by SEQ ID NO:22, SEQID NO:23, SEQ ID NO:24 or SEQ ID NO:73, in which 1 to 15 (preferably 1to 10, more preferably 1 to 5, and most preferably 1 to 3) amino acidsare replaced by other amino acids;

[0353] (ix) amino acid sequences in combination of (v) to (viii) above;etc.

[0354] The human precursor peptide G having the amino acid sequencerepresented by SEQ ID NO:19 is the peptide wherein a secretory signalsequence is removed from the human precursor peptide H having the aminoacid sequence represented by SEQ ID NO:22.

[0355] The human precursor peptide G having the amino acid sequencerepresented by SEQ ID NO:20 is the peptide wherein a secretory signalsequence is removed from the human precursor peptide H having the aminoacid sequence represented by SEQ ID NO:23.

[0356] The human precursor peptide G having the amino acid sequencerepresented by SEQ ID NO:21 is the peptide wherein a secretory signalsequence is removed from the human precursor peptide H having the aminoacid sequence represented by SEQ ID NO:24.

[0357] The human precursor peptide G having the amino acid sequencerepresented by SEQ ID NO:72 is the peptide wherein a secretory signalsequence is removed from the human precursor peptide H having the aminoacid sequence represented by SEQ ID NO:73.

[0358] The precursor peptide of the present invention may have similaractivities to those of the peptide of the present invention.

[0359] The protein (human GPR7) having the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:49, the protein (rat TGR26) having the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:59, the protein (human GPR8) having the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:84, the protein (bovine GPR7) having the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:86, the protein (bovine GPR8) having the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:88 may be any protein derived from any cells of human or otherwarm-blooded animals, e.g., guinea pigs, rats, mice, chicken, rabbits,swine, sheep, bovine, monkeys, etc. (e.g., retina cells, liver cells,splenocytes, nerve cells, glial cells, β cells of pancreas, bone marrowcells, mesangial cells, Langerhans' cells, epidermic cells, epithelialcells, endothelial cells, fibroblasts, fibrocytes, myocytes, fat cells,immune cells (e.g., macrophages, T cells, B cells, natural killer cells,mast cells, neutrophils, basophils, eosinophils, monocytes),megakaryocyte, synovial cells, chondrocytes, bone cells, osteoblasts,osteoclasts, mammary gland cells, hepatocytes or interstitial cells, thecorresponding precursor cells, stem cells, cancer cells, etc., of thesecells), or any tissues where such cells are present, e.g., brain or anyregion of the brain (e.g., retina, olfactory bulb, amygdaloid nucleus,basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex,medulla oblongata, cerebellum), spinal cord, hypophysis, stomach,pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow,adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g., largeintestine and small intestine), blood vessel, heart, thymus, spleen,submandibular gland, peripheral blood, prostate, testis, ovary,placenta, uterus, bone, joint, skeletal muscle, etc.; or hemocyte typecells or their cultured cells (e.g., MEL, M1, CTLL-2, HT-2, WEHI-3,HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1,Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL,JK-1, CMK, KO-812, MEG-01, etc.). The protein may also be a syntheticprotein.

[0360] The amino acid sequence which has substantially the same aminoacid sequence as that represented by SEQ ID NO:49 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, and more preferably at least about 90% homology, to theamino acid sequence represented by SEQ ID NO:49.

[0361] In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:49, include, in addition to the amino acid sequence described above:

[0362] (i) the amino acid sequence represented by SEQ ID NO:49, in which1 to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably1 to 3) amino acids are deleted;

[0363] (ii) the amino acid sequence represented by SEQ ID NO:49, towhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are added;

[0364] (iii) the amino acid sequence represented by SEQ ID NO:49, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are inserted;

[0365] (iv) the amino acid sequence represented by S SEQ ID NO:49, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are replaced by other amino acids;

[0366] (v) amino acid sequences in combination of (i) to (iv) above;etc.

[0367] The amino acid sequence which has substantially the same aminoacid sequence as that represented by SEQ ID NO:59 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, and more preferably at least about 90% homology, to theamino acid sequence represented by SEQ ID NO:59.

[0368] In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:59, include, in addition to the amino acid sequence described above:

[0369] (i) the amino acid sequence represented by SEQ ID NO:59, in which1 to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably1 to 3) amino acids are deleted;

[0370] (ii) the amino acid sequence represented by SEQ ID NO:59, towhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are added;

[0371] (iii) the amino acid sequence represented by SEQ ID NO:59, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are inserted;

[0372] (iv) the amino acid sequence represented by S SEQ ID NO:59, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are replaced by other amino acids;

[0373] (v) amino acid sequences in combination of (i) to (iv) above;etc.

[0374] The amino acid sequence which has substantially the same aminoacid sequence as that represented by SEQ ID NO:84 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, and more preferably at least about 90% homology, to theamino acid sequence represented by SEQ ID NO:84.

[0375] In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:84, include, in addition to the amino acid sequence described above:

[0376] (i) the amino acid sequence represented by SEQ ID NO:84, in which1 to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably1 to 3) amino acids are deleted;

[0377] (ii) the amino acid sequence represented by SEQ ID NO:84, towhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are added;

[0378] (iii) the amino acid sequence represented by SEQ ID NO:84, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are inserted;

[0379] (iv) the amino acid sequence represented by S SEQ ID NO:84, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are replaced by other amino acids;

[0380] (v) amino acid sequences in combination of (i) to (iv) above;etc.

[0381] The amino acid sequence which has substantially the same aminoacid sequence as that represented by SEQ ID NO:86 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to the amino acid sequencerepresented by SEQ ID NO:86.

[0382] In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:86, include, in addition to the amino acid sequence described above:

[0383] (i) the amino acid sequence represented by SEQ ID NO:86, in which1 to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably1 to 3) amino acids are deleted;

[0384] (ii) the amino acid sequence represented by SEQ ID NO:86, towhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are added;

[0385] (iii) the amino acid sequence represented by SEQ ID NO:86, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are inserted;

[0386] (iv) the amino acid sequence represented by S SEQ ID NO:86, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are replaced by other amino acids;

[0387] (v) amino acid sequences in combination of (i) to (iv) above;etc.

[0388] The amino acid sequence which has substantially the same aminoacid sequence as that represented by SEQ ID NO:88 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to the amino acid sequencerepresented by SEQ ID NO:88.

[0389] In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:88, include, in addition to the amino acid sequence described above:

[0390] (i) the amino acid sequence represented by SEQ ID NO:88, in which1 to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably1 to 3) amino acids are deleted;

[0391] (ii) the amino acid sequence represented by SEQ ID NO:88, towhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are added;

[0392] (iii) the amino acid sequence represented by SEQ ID NO:88, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are inserted;

[0393] (iv) the amino acid sequence represented by S SEQ ID NO:88, inwhich 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and mostpreferably 1 to 3) amino acids are replaced by other amino acids;

[0394] (v) amino acid sequences in combination of (i) to (iv) above;etc.

[0395] The partial peptide of human GPR7, rat TGR26, human GPR8, bovineGPR7 or bovine GPR8 (hereinafter merely referred to as GPR7collectively) may be any peptide, so long as it is a partial peptideusable in the methods for screening pharmaceuticals, etc. laterdescribed, but, a partial peptide capable of binding to the peptide ofthe present invention, a partial peptide containing the correspondingamino acid sequence in the area outside the cell membrane, etc. arepreferably employed.

[0396] The peptide of the present invention, its partial peptide or itsprecursor peptide, especially the peptide of the present invention alsoincludes a peptide wherein the N-terminal amino acid residue isbrominated. Preferred examples of the N-terminal amino acid residue aretryptophan residue (Trp), etc.

[0397] Specifically, a peptide containing the amino acid sequenceselected from SEQ ID NO:1 to SEQ ID NO:12, SEQ ID NO:19 to SEQ ID NO:21,SEQ ID NO:66 to SEQ ID NO:69 and SEQ ID NO:72, etc., wherein theN-terminal tryptophan residue (Trp) is brominated, is employed. Amongthese peptides, preferably employed is a peptide containing the aminoacid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:66 or SEQ ID NO:67, whereinthe N-terminal tryptophan residue (Trp) is brominated. The position tobe brominated is not particularly limited but the tryptophan residue(Trp) at the 6-position is preferred.

[0398] More specifically, a peptide containing the amino acid sequenceselected from SEQ ID NO:1 to SEQ ID NO:12, SEQ ID NO:19 to SEQ ID NO:21,SEQ ID NO:66 to SEQ ID NO:69 and SEQ ID NO:72, wherein the N-terminaltryptophan residue (Trp) is 6-brominated, is preferably employed. Amongthem, preferably employed is a peptide containing the amino acidsequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ. ID NO:66 or SEQ ID NO:67, whereinthe N-terminal tryptophan residue (Trp) is 6-brominated.

[0399] The peptide of the present invention, its partial peptide or itsprecursor peptide (hereinafter sometimes merely referred to as thepeptide of the present invention), and GPR7 or its partial peptide(hereinafter sometimes merely referred to as GPR7) are represented inaccordance with the conventional way of describing peptides, that is,the N-terminus (amino terminus) at the left hand and the C-terminus(carboxyl terminus) at the right hand.

[0400] In the peptide of the present invention or GPR7, the C-terminusmay be any of a carboxyl group (—COOH), a carboxylate (—COO⁻), an amide(—CONH₂) or an ester (—COOR).

[0401] Examples of the ester group shown by R include a C₁₋₆ alkyl groupsuch as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.; a C₃₋₈cycloalkyl group such as cyclopentyl, cyclohexyl, etc.; a C₆₋₁₂ arylgroup such as phenyl, α-naphthyl, etc.; a C₇₋₁₄ aralkyl group such as aphenyl-C₁₋₂-alkyl group, e.g., benzyl, phenethyl, etc., or anα-naphthyl-C₁₋₂-alkyl group such as α-naphthylmethyl, etc.; and thelike. In addition, pivaloyloxymethyl or the like, which is used widelyas an ester for oral administration, may also be used.

[0402] Where the peptide of the present invention or GPR7 contains acarboxyl group (or a carboxylate) at a position other than theC-terminus, it may be amidated or esterified and such an amide or esteris also included within the peptide of the present invention. The estergroup may be the same group as that described with respect to theC-terminus described above.

[0403] Furthermore, the peptide of the present invention or GPR7includes peptides, wherein the amino group at the N-terminal amino acidresidue (e.g., methionine residue) is protected with a protecting group(e.g., a C₁₋₆ acyl group such as a C₂₋₆ alkanoyl group, e.g., formylgroup, acetyl group, etc.); those wherein the N-terminal region iscleaved in vivo and the glutamyl group thus formed is pyroglutaminated;those wherein a substituent (e.g., —OH, —SH, amino group, imidazolegroup, indole group, guanidino group, etc.) on the side chain of anamino acid in the molecule is protected with a suitable protecting group(e.g., a C₁₋₆ acyl group such as a C₂₋₆ alkanoyl group, e.g., formylgroup, acetyl group, etc.), or conjugated proteins such as glycoproteinsbound to sugar chains.

[0404] For salts of the peptide of the present invention or GPR7,preferred are salts with physiologically acceptable acids (e.g.,inorganic acids or organic acids) or bases (e.g., alkali metal salts),etc., especially physiologically acceptable acid addition salts.Examples of such salts include salts with, for example, inorganic acids(e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuricacid); salts with organic acids (e.g., acetic acid, formic acid,propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid,citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonicacid, benzenesulfonic acid) and the like. Hereinafter, the peptide ofthe present invention or GPR7 is used to include these salts as well.

[0405] The peptide of the present invention or GPR7 may be manufacturedby a publicly known method used to purify a peptide from human or otherwarm-blooded animal cells or tissues described above, or by culturing atransformant that contains the DNA encoding the peptide, as will belater described. Furthermore, the peptide of the present invention orGPR7 may also be manufactured by the methods for synthesizing peptidesor by modifications thereof, which will also be described hereinafter.

[0406] Where the peptide of the present invention or GPR7 ismanufactured from human or mammalian tissues or cells, human ormammalian tissues or cells are homogenized, then extracted with an acidor the like, and the extract is isolated and purified by a combinationof chromatography techniques such as reverse phase chromatography, ionexchange chromatography, and the like.

[0407] To synthesize the peptide of the present invention or GPR7 oramides thereof, commercially available resins that are used for peptidesynthesis may be used. Examples of such resins include chloromethylresin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAMresin, 4-hydroxymethylmehtylphenyl acetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenylhydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin, etc. Usingthese resins, amino acids in which α-amino groups and functional groupson the side chains are appropriately protected are condensed on theresin in the order of the sequence of the objective peptide according tovarious condensation methods publicly known in the art. At the end ofthe reaction, the peptide is cut out from the resin and at the sametime, the protecting groups are removed. Then, intramolecular disulfidebond-forming reaction is performed in a highly diluted solution toobtain the objective peptide of the present invention or GPR7, or amidesthereof.

[0408] For condensation of the protected amino acids described above, avariety of activation reagents for peptide synthesis may be used, andcarbodiimides are particularly preferable. Examples of suchcarbodiimides include DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide, etc. For activation bythese reagents, the protected amino acids in combination with aracemization inhibitor (e.g., HOBt, HOOBt) are added directly to theresin, or the protected amino acids are previously activated in the formof symmetric acid anhydrides, HOBt esters or HOOBt esters, followed byadding the thus activated protected amino acids to the resin.

[0409] Solvents suitable for use to activate the protected amino acidsor condense with the resin may be chosen from solvents known to beusable for peptide condensation reactions. Examples of such solvents areacid amides such as N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpyrrolidone, etc.; halogenated hydrocarbons such as methylenechloride, chloroform, etc.; alcohols such as trifluoroethanol, etc.;sulfoxides such as dimethylsulfoxide, etc.; ethers such as pyridine,dioxan, tetrahydrofuran, etc.; nitriles such as acetonitrile,propionitrile, etc.; esters such as methyl acetate, ethyl acetate, etc.;and appropriate mixtures of these solvents. The reaction temperature isappropriately chosen from the range known to be applicable to peptidebinding reactions and is usually selected in the range of approximately−20° C. to 50° C. The activated amino acid derivatives are usedgenerally in an excess of 1.5 to 4 times. The condensation is examinedby a test using the ninhydrin reaction; when the condensation isinsufficient, the condensation can be completed by repeating thecondensation reaction without removal of the protecting groups. When thecondensation is yet insufficient even after repeating the reaction,unreacted amino acids are acetylated with acetic anhydride oracetylimidazole so as not to affect the following reactions.

[0410] Examples of the protecting groups for amino groups of thestarting compounds include Z, Boc, t-pentyloxycarbonyl,isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z,adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl,2-nitrophenylsulphenyl, diphenylphosphinothioyl, Fmoc, etc.

[0411] A carboxyl group can be protected by, e.g., alkyl esterification(e.g., esterification of linear, branched or cyclic alkyl moiety such asmethyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl, etc.), aralkyl esterification(e.g., esterification in the form of benzyl ester, 4-nitrobenzyl ester,4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, etc.),phenacyl esterification, benzyloxycarbonyl hydrazidation,t-butoxycarbonyl hydrazidation, trityl hydrazidation, or the like.

[0412] The hydroxyl group of serine can be protected through, forexample, its esterification or etherification. Examples of groupsappropriately used for the esterification include a lower alkanoylgroup, such as acetyl group, an aroyl group such as benzoyl group, and agroup derived from carbonic acid such as benzyloxycarbonyl group,ethoxycarbonyl group, etc. Examples of a group appropriately used forthe etherification include benzyl group, tetrahydropyranyl group,t-butyl group, etc.

[0413] Examples of groups for protecting the phenolic hydroxyl group oftyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, t-butyl, etc.

[0414] Examples of groups used to protect the imidazole moiety ofhistidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP,benzyloxymethyl, Bum, Boc, Trt, Fmoc, etc.

[0415] Examples of the activated carboxyl groups in the startingcompounds include the corresponding acid anhydrides, azides, activatedesters (esters with alcohols (e.g., pentachlorophenol,2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol,p-nitrophenol, HONB, N-hydroxysuccimide, N-hydroxyphthalimide, HOBt)).As the activated amino acids, in which the amino groups are activated inthe starting material, the corresponding phosphoric amides are employed.

[0416] To eliminate (split off) the protecting groups, there are usedcatalytic reduction under hydrogen gas flow in the presence of acatalyst such as Pd-black or Pd-carbon; an acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethane-sulfonic acidor trifluoroacetic acid, or a mixture solution of these acids; atreatment with a base such as diisopropylethylamine, triethylamine,piperidine or piperazine; and reduction with sodium in liquid ammonia.The elimination of the protecting group by the acid treatment describedabove is carried out generally at a temperature of approximately −20° C.to 40° C. In the acid treatment, it is efficient to add a cationscavenger such as anisole, phenol, thioanisole, m-cresol, p-cresol,dimethylsulfide, 1,4-butanedithiol or 1,2-ethanedithiol. Furthermore,2,4-dinitrophenyl group known as the protecting group for the imidazoleof histidine is removed by a treatment with thiophenol. Formyl groupused as the protecting group of the indole of tryptophan is eliminatedby the aforesaid acid treatment in the presence of 1,2-ethanedithiol or1,4-butanedithiol, as well as by a treatment with an alkali such as adilute sodium hydroxide solution, dilute ammonia, etc.

[0417] Protection of functional groups that should not be involved inthe reaction of the starting materials, protecting groups, eliminationof the protecting groups and activation of functional groups involved inthe reaction may be appropriately selected from publicly known groupsand publicly known means.

[0418] In another method for obtaining the amides of the peptide of thepresent invention or GPR7, for example, the α-carboxyl group of thecarboxy terminal amino acid is first protected by amidation; the peptidechain is then extended from the amino group side to a desired length.Thereafter, a peptide in which only the protecting group of theN-terminal α-amino group in the peptide chain has been eliminated fromthe peptide and a peptide in which only the protecting group of theC-terminal carboxyl group has been eliminated are prepared. The twopeptides are condensed in a mixture of the solvents described above. Thedetails of the condensation reaction are the same as described above.After the protected peptide obtained by the condensation is purified,all the protecting groups are eliminated by the method described aboveto give the desired crude peptide. This crude peptide is purified byvarious known purification means. Lyophilization of the major fractiongives the amides of the desired peptide of the present invention orGPR7.

[0419] To prepare the esterified form of the peptide of the presentinvention or GPR7, for example, the α-carboxyl group of the carboxyterminal amino acid is condensed with a desired alcohol to prepare theamino acid ester, which is followed by procedure similar to thepreparation of the amidated form of the peptide of the present inventionor GPR7 to give the desired ester form of the peptide of the presentinvention or GPR7.

[0420] The partial peptide of the peptide of the present invention orGPR7 can be manufactured by publicly known methods for peptidesynthesis, or the partial peptide of GPR7 can be manufactured bycleaving GPR7 with an appropriate peptidase. For the methods for peptidesynthesis, for example, either solid phase synthesis or liquid phasesynthesis may be used. That is, the partial peptide or amino acids thatcan construct the peptide of the present invention or the partialpeptide of GPR7 are condensed with the remaining part. Where the productcontains protecting groups, these protecting groups are removed to givethe desired peptide. Publicly known methods for condensation andelimination of the protecting groups are described in (1)-(5) below.

[0421] (1) M. Bodanszky & M. A. Ondetti: Peptide Synthesis, IntersciencePublishers, New York (1966)

[0422] (2) Schroeder & Luebke: The Peptide, Academic Press, New York(1965)

[0423] (3) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken(Basics and experiments of peptide synthesis), published by Maruzen Co.(1975)

[0424] (4) Haruaki Yajima & Shunpei Sakakibara: Seikagaku Jikken Koza(Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry ofProteins) IV, 205 (1977)

[0425] (5) Haruaki Yajima, ed.: Zoku Iyakuhin no Kaihatsu (A sequel toDevelopment of Pharmaceuticals), Vol. 14, Peptide Synthesis, publishedby Hirokawa Shoten

[0426] After completion of the reaction, the peptide of the presentinvention, GPR7 or a partial peptide thereof may be purified andisolated by a combination of conventional purification methods such assolvent extraction, distillation, column chromatography, liquidchromatography and recrystallization to give the partial peptide of thepresent invention. When the peptide of the present invention or thepartial peptide of GPR7 obtained by the above methods is in a free form,the peptide can be converted into an appropriate salt by a publiclyknown method; when the product is obtained in the form of a salt, it canbe converted into a free form or other salts by a publicly known methodor its modification.

[0427] The polynucleotide encoding the peptide of the present inventionor GPR7 may be any polynucleotide so long as it contains the basesequence (DNA or RNA, preferably DNA) encoding the peptide of thepresent invention or GPR7 described above. Such a polynucleotide mayalso be any one of DNA encoding the peptide of the present invention orGPR7, RNA such as mRNA, etc., and may be double-stranded orsingle-stranded. Where the polynucleotide is double-stranded, it may bedouble-stranded DNA, double-stranded RNA or DNA:RNA hybrid. Where thepolynucleotide is single-stranded, it may be a sense strand (i.e., acoding strand) or an antisense strand (i.e., a non-coding strand).

[0428] Using the polynucleotide encoding the peptide of the presentinvention or GPR7, mRNA of the peptide of the present invention or GPR7can be quantified by, for example, the publicly known method publishedin separate volume of Jikken Igaku 15 (7) “New PCR and its application”(1997), or by its modifications.

[0429] The DNA encoding the peptide of the present invention or GPR7 maybe any DNA, as long as it contains a base sequence encoding the peptideof the present invention or GPR7 described above. The DNA may also beany of genomic DNA, genomic DNA library, cDNA derived from the cells andtissues described above, cDNA library derived from the cells and tissuesdescribed above and synthetic DNA.

[0430] The vector to be used for the library may be any ofbacteriophage, plasmid, cosmid and phagemid. The DNA may also bedirectly amplified by reverse transcriptase polymerase chain reaction(hereinafter abbreviated as RT-PCR) using the total RNA or mRNA fractionprepared from the cells and tissues described above.

[0431] The DNA encoding the peptide of the present invention may be anyDNA, so long as it is a DNA having a base sequence hybridizable to thebase sequence represented by any sequence identification number of SEQID NO:25 to SEQ ID NO:42 and SEQ ID NO:74 to SEQ ID NO:79 under highlystringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of the peptide of the presentinvention.

[0432] Specific examples of the DNA hybridizable to the base sequencerepresented by any sequence identification number of SEQ ID NO:25 to SEQID NO:42 and SEQ ID NO:74 to SEQ ID NO:79 under highly stringentconditions include DNAs containing a base sequence having at least about70% homology, preferably at least about 80% homology, more preferably atleast about 90% homology and the most preferably at least about 95%homology, to the base sequence represented by any sequenceidentification number of SEQ ID NO:25 to SEQ ID NO:42 and SEQ ID NO:74to SEQ ID NO:79.

[0433] The hybridization can be carried out by publicly known methods orby modifications of these methods, for example, according to the methoddescribed in Molecular Cloning, 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989). A commercially available library may also beused according to the instructions of the attached manufacturer'sprotocol. Preferably, the hybridization can be carried out under highlystringent conditions.

[0434] The highly stringent conditions used herein are, for example,those in a sodium concentration at about 19 to 40 mM, preferably about19 to 20 mM at a temperature of about 50 to 70° C., preferably about 60to 65° C. In particular, hybridization conditions in a sodiumconcentration of about 19 mM at a temperature of about 65° C. are mostpreferred.

[0435] More specifically,

[0436] (i) for the DNA encoding human peptide A containing the aminoacid sequence represented by SEQ ID NO:1, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:25, etc.;

[0437] (ii) for the DNA encoding mouse peptide A containing the aminoacid sequence represented by SEQ ID NO:2, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:26, etc.;

[0438] (iii) for the DNA encoding rat peptide A containing the aminoacid sequence represented by SEQ ID NO:3, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:27, etc.;

[0439] (iv) for the DNA encoding human peptide B containing the aminoacid sequence represented by SEQ ID NO:4, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:28, etc.;

[0440] (v) for the DNA encoding mouse peptide B containing the aminoacid sequence represented by SEQ ID NO:5, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:29, etc.;

[0441] (vi) for the DNA encoding rat peptide B containing the amino acidsequence represented by SEQ ID NO:6, there are employed a DNA containingthe base sequence represented by SEQ ID NO:30, etc.;

[0442] (vii) for the DNA encoding human peptide C containing the aminoacid sequence represented by SEQ ID NO:7, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:31, etc.;

[0443] (viii) for the DNA encoding human peptide D containing the aminoacid sequence represented by SEQ ID NO:8, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:32, etc.;

[0444] (ix) for the DNA encoding mouse peptide C containing the aminoacid sequence represented by SEQ ID NO:9, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:33, etc.;

[0445] (x) for the DNA encoding mouse peptide D containing the aminoacid sequence represented by SEQ ID NO:10, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:34, etc.;

[0446] (xi) for the DNA encoding rat peptide C containing the amino acidsequence represented by SEQ ID NO:11, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:35, etc.;

[0447] (xii) for the DNA encoding rat peptide D containing the aminoacid sequence represented by SEQ ID NO:12, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:36, etc.;

[0448] (xiii) for the DNA encoding human peptide E containing the aminoacid sequence represented by SEQ ID NO:13, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:37, etc.;

[0449] (xiv) for the DNA encoding mouse peptide E containing the aminoacid sequence represented by SEQ ID NO:14, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:38, etc.;

[0450] (xv) for the DNA encoding rat peptide E containing the amino acidsequence represented by SEQ ID NO:15, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:39, etc.;

[0451] (xvi) for the DNA encoding human peptide F containing the aminoacid sequence represented by SEQ ID NO:16, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:40, etc.;

[0452] (xvii) for the DNA encoding mouse peptide F containing the aminoacid sequence represented by SEQ ID NO:17, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:41, etc.;

[0453] (xviii) for the DNA encoding rat peptide F containing the aminoacid sequence represented by SEQ ID NO:18, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:42, etc.;

[0454] (xix) for the DNA encoding bovine peptide A containing the aminoacid sequence represented by SEQ ID NO:66, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:74, etc.;

[0455] (xx) for the DNA encoding bovine peptide B containing the aminoacid sequence represented by SEQ ID NO:67, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:75, etc.;

[0456] (xxi) for the DNA encoding bovine peptide C containing the aminoacid sequence represented by SEQ ID NO:68, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:76, etc.;

[0457] (xxii) for the DNA encoding bovine peptide D containing the aminoacid sequence represented by SEQ ID NO:69, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:77, etc.;

[0458] (xxiii) for the DNA encoding bovine peptide E containing theamino acid sequence represented by SEQ ID NO:70, there are employed aDNA containing the base sequence represented by SEQ ID NO:78, etc.;

[0459] (xxvi) for the DNA encoding bovine peptide F containing the aminoacid sequence represented by SEQ ID NO:71, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:79; etc.

[0460] The DNA encoding the partial peptide of the present invention maybe any DNA, as long as it contains a base sequence encoding the partialpeptide of the present invention described above. The DNA may also beany of genomic DNA, genomic DNA library, cDNA derived from the cells andtissues described above, cDNA library derived from the cells and tissuesdescribed above and synthetic DNA.

[0461] The DNA encoding the partial peptide of the present invention is,for example, a DNA having a partial base sequence of the DNA having thebase sequence represented by any sequence identification number of SEQID NO:25 to SEQ ID NO:42 and SEQ ID NO:74 to SEQ ID NO:79, or a DNAhaving a base sequence hybridizable to the base sequence represented byany sequence identification number of SEQ ID NO:25 to SEQ ID NO:42 andSEQ ID NO:74 to SEQ ID NO:79 under highly stringent conditions andencoding a peptide having the activities substantially equivalent tothose of the peptide of the present invention.

[0462] The DNA hybridizable to the base sequence represented by anysequence identification number of SEQ ID NO:25 to SEQ ID NO:42 and SEQID NO:74 to SEQ ID NO:79 has the same significance as described above.

[0463] With respect to the hybridization under high stringentconditions, the same procedures as described above apply.

[0464] The DNA encoding the precursor peptide of the present inventionmay be any DNA, so long as it is a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:43 or SEQ IDNO:46 under-highly stringent conditions and encoding a peptide havingthe activities substantially equivalent to those of the precursor of thepresent invention.

[0465] The DNA hybridizable to the base sequence represented by SEQ IDNO:43 or SEQ ID NO:46 under highly stringent conditions includes a DNAcontaining a base sequence having at least about 70% homology,preferably at least about 80% homology, more preferably at least about90% homology, and most preferably at least about 95% homology, to a basesequence represented by any sequence identification number of SEQ IDNO:43 or SEQ ID NO:46.

[0466] With respect to the hybridization under high stringentconditions, the same procedures the same conditions as described aboveapply.

[0467] More specifically,

[0468] (i) for the DNA encoding human GPR7 ligand precursor G containingthe amino acid sequence represented by SEQ ID NO:19, there are employeda DNA containing the base sequence represented by SEQ ID NO:43, etc.;

[0469] (ii) for the DNA encoding mouse GPR7 ligand precursor Gcontaining the amino acid sequence represented by SEQ ID NO:20, thereare employed a DNA containing the base sequence represented by SEQ IDNO:44, etc.;

[0470] (iii) for the DNA encoding rat GPR7 ligand precursor G containingthe amino acid sequence represented by SEQ ID NO:21, there are employeda DNA containing the base sequence represented by SEQ ID NO:45, etc.;

[0471] (iv) for the DNA encoding bovine GPR7 ligand precursor Gcontaining the amino acid sequence represented by SEQ ID NO:72, thereare employed a DNA containing the base sequence represented by SEQ IDNO:80, etc.;

[0472] (v) for the DNA encoding mouse GPR7 ligand precursor H containingthe amino acid sequence represented by SEQ ID NO:22, there are employeda DNA containing the base sequence represented by SEQ ID NO:46, etc.;

[0473] (vi) for the DNA encoding mouse GPR7 ligand precursor Hcontaining the amino acid sequence represented by SEQ ID NO:23, thereare employed a DNA containing the base sequence represented by SEQ IDNO:47, etc.;

[0474] (vii) for the DNA encoding rat GPR7 ligand precursor H containingthe amino acid sequence represented by SEQ ID NO:24, there are employeda DNA containing the base sequence represented by SEQ ID NO:48, etc.;

[0475] (viii) for the DNA encoding bovine GPR7 ligand precursor Hcontaining the amino acid sequence represented by SEQ ID NO:73, thereare employed a DNA containing the base sequence represented by SEQ IDNO:81, etc.

[0476] The polynucleotide comprising a part of the base sequence of theDNA encoding the peptide or partial peptide of the present invention, ora part of the base sequence complementary to the DNA is used to mean toembrace not only the DNA encoding the partial peptide of the presentinvention but also RNA.

[0477] According to the present invention, antisense polynucleotides(nucleic acids) that can inhibit the replication or expression of genesfor the peptide of the present invention can be designed and synthesizedbased on the base sequence information of the cloned or determined DNAencoding the peptide of the present invention. Such a polynucleotide(nucleic acid) is capable of hybridizing to RNA of genes for the peptideof the present invention to inhibit the synthesis or function of saidRNA or capable of modulating or controlling the expression of genes forthe peptide of the present invention via interaction with RNA associatedwith the peptide of the present invention. Polynucleotides complementaryto the selected sequences of RNA associated with the peptide of thepresent invention and polynucleotides specifically hybridizable to theRNA associated with the peptide of the present invention are useful inmodulating or controlling the expression of genes for the peptide of thepresent invention in vivo and in vitro, and useful for the treatment ordiagnosis of diseases, etc. The term “corresponding” is used to meanhomologous to or complementary to a particular sequence of thenucleotide, base sequence or nucleic acid, including the genes. The term“corresponding” between nucleotides, base sequences or nucleic acids andpeptides (proteins) usually refer to amino acids of a peptide (protein)under the order derived from the sequence of nucleotides (nucleic acids)or their complements. In the genes for the peptide of the presentinvention, the 5′ end hairpin loop, 5′ end 6-base-pair repeats, 5′ enduntranslated region, polypeptide translation initiation codon, proteincoding region, ORF translation initiation codon, 3′ end untranslatedregion, 3′ end palindrome region, and 3′ end hairpin loop, may beselected as preferred target regions, though any other region may beselected as a target in the genes for the peptide of the presentinvention.

[0478] The relationship between the targeted nucleic acids and thepolynucleotides complementary to at least a part of the target,specifically the relationship between the target and the polynucleotideshybridizable to the target, can be denoted to be “antisense”. Examplesof the antisense polynucleotides include polynucleotides containing2-deoxy-D-ribose, polynucleotides containing D-ribose, any other type ofpolynucleotides which are N-glycosides of a purine or pyrimidine base,or other polymers containing non-nucleotide backbones (e.g., proteinnucleic acids and synthetic sequence-specific nucleic acid polymerscommercially available) or other polymers containing nonstandardlinkages (provided that the polymers contain nucleotides having such aconfiguration that allows base pairing or base stacking, as is found inDNA or RNA), etc. The antisense polynucleotides may be double-strandedDNA, single-stranded DNA, single-stranded RNA or a DNA:RNA hybrid, andmay further include unmodified polynucleotides (or unmodifiedoligonucleotides), those with publicly known types of modifications, forexample, those with labels known in the art, those with caps, methylatedpolynucleotides, those with substitution of one or more naturallyoccurring nucleotides by their analogue, those with intramolecularmodifications of nucleotides such as those with uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoramidates,carbamates, etc.) and those with charged linkages or sulfur-containinglinkages (e.g., phosphorothioates, phosphorodithioates, etc.), thosehaving side chain groups such as proteins (nucleases, nucleaseinhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.),saccharides (e.g., monosaccharides, etc.), those with intercalators(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,metals, radioactive metals, boron, oxidative metals, etc.), thosecontaining alkylating agents, those with modified linkages (e.g., aanomeric nucleic acids, etc.), and the like. Herein the terms“nucleoside”, “nucleotide” and “nucleic acid” are used to refer tomoieties that contain not only the purine and pyrimidine bases, but alsoother heterocyclic bases, which have been modified. Such modificationsmay include methylated purines and pyrimidines, acylated purines andpyrimidines and other heterocyclic rings. Modified nucleotides andmodified nucleotides also include modifications on the sugar moiety,wherein, for example, one or more hydroxyl groups may optionally besubstituted with a halogen atom(s), an aliphatic group(s), etc., or maybe converted into the corresponding functional groups such as ethers,amines, or the like.

[0479] The antisense polynucleotide (nucleic acid) of the presentinvention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specificexamples of the modified nucleic acid are, but not limited to, sulfurand thiophosphate derivatives of nucleic acids and those resistant todegradation of polynucleoside amides or oligonucleoside amides. Theantisense nucleic acids of the present invention can be modifiedpreferably based on the following design, that is, by increasing theintracellular stability of the antisense nucleic acid, increasing thecellular permeability of the antisense nucleic acid, increasing theaffinity of the nucleic acid to the targeted sense strand to a higherlevel, or minimizing the toxicity, if any, of the antisense nucleicacid.

[0480] Many of such modifications are known in the art, as disclosed inJ. Kawakami, et al., Pharm. Tech. Japan, Vol. 8, pp. 247, 1992; Vol. 8,pp. 395, 1992; S. T. Crooke, et al. ed., Antisense Research andApplications, CRC Press, 1993; etc.

[0481] The antisense nucleic acid of the present invention may containaltered or modified sugars, bases or linkages. The antisense nucleicacid may also be provided in a specialized form such as liposomes,microspheres, or may be applied to gene therapy, or may be provided incombination with attached moieties. Such attached moieties includepolycations such as polylysine that act as charge neutralizers of thephosphate backbone, or hydrophobic moieties such as lipids (e.g.,phospholipids, cholesterols, etc.) that enhance the interaction withcell membranes or increase uptake of the nucleic acid. Preferredexamples of the lipids to be attached are cholesterols or derivativesthereof (e.g., cholesteryl chloroformate, cholic acid, etc.). Thesemoieties may be attached to the nucleic acid at the 3′ or 5′ endsthereof and may also be attached thereto through a base, sugar, orintramolecular nucleoside linkage. Other moieties may be capping groupsspecifically placed at the 3′ or 5′ ends of the nucleic acid to preventdegradation by nucleases such as exonuclease, RNase, etc. Such cappinggroups include, but are not limited to, hydroxyl protecting groups knownin the art, including glycols such as polyethylene glycol, tetraethyleneglycol and the like.

[0482] The inhibitory action of the antisense nucleic acid can beexamined using the transformant of the present invention, the geneexpression system of the present invention in vivo and in vitro, or thetranslation system of the peptide of the present invention in vivo andin vitro. The nucleic acid can be applied to cells by a variety ofpublicly known methods.

[0483] The DNA encoding human GPR7 may be any DNA, as far as it is a DNAcontaining the base sequence represented by, e.g., SEQ ID NO:50, a DNAhaving a base sequence hybridizable to the base sequence represented bySEQ ID NO:50 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of human GPR7having the base sequence represented by SEQ ID NO:49, or the like.

[0484] The DNA hybridizable to the base sequence represented by SEQ IDNO:50 under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:50, or the like.

[0485] The DNA encoding rat TGR26 may be any DNA, as far as it is a DNAcontaining the base sequence represented by, e.g., SEQ ID NO:60, a DNAhaving a base sequence hybridizable to the base sequence represented bySEQ ID NO:60 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of rat TGR26having the base sequence represented by SEQ ID NO:59, or the like.

[0486] The DNA hybridizable to the base sequence represented by SEQ IDNO:60 under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:60, or the like.

[0487] The DNA encoding human GPR8 may be any DNA, as far as it is a DNAcontaining the base sequence represented by, e.g., SEQ ID NO:85, a DNAhaving a base sequence hybridizable to the base sequence represented bySEQ ID NO:85 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of human GPR8having the base sequence represented by SEQ ID NO:66, or the like.

[0488] The DNA hybridizable to the base sequence represented by SEQ IDNO:85 under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:85, or the like.

[0489] The DNA encoding bovine GPR7 may be any DNA, as far as it is aDNA containing the base sequence represented by, e.g., SEQ ID NO:87, aDNA having a base sequence hybridizable to the base sequence representedby SEQ ID NO:87 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of bovine GPR7having the base sequence represented by SEQ ID NO:86, or the like.

[0490] The DNA hybridizable to the base sequence represented by SEQ IDNO:87 under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:87, or the like.

[0491] The DNA encoding bovine GPR8 may be any DNA, as far as it is aDNA containing the base sequence represented by, e.g., SEQ ID NO:89, aDNA having a base sequence hybridizable to the base sequence representedby SEQ ID NO:89 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of bovine GPR8having the base sequence represented by SEQ ID NO:88, or the like.

[0492] The DNA hybridizable to the base sequence represented by SEQ IDNO:89 under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:89, or the like.

[0493] The hybridization can be carried out by publicly known methods orby modifications of these methods, for example, according to the methoddescribed in Molecular Cloning, 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989), etc. A commercially available library may alsobe used according to the instructions of the attached manufacturer'sprotocol. Preferably, the hybridization can be carried out under highlystringent conditions.

[0494] The highly stringent conditions used herein are, for example,those in a sodium concentration at about 19 to 40 mM, preferably about19 to 20 mM at a temperature of about 50 to 70° C., preferably about 60to 65° C. In particular, hybridization conditions in a sodiumconcentration of about 19 mM at a temperature of about 65° C. are mostpreferred.

[0495] More specifically, for the DNA encoding human GPR7 containing theamino acid sequence represented by SEQ ID NO:49, there are employed aDNA containing the base sequence represented by SEQ ID NO:50, etc.; forthe DNA encoding rat TGR26 containing the amino acid sequencerepresented by SEQ ID NO:59, there are employed a DNA containing thebase sequence represented by SEQ ID NO:60, etc.; for the DNA encodinghuman GPR8 containing the amino acid sequence represented by SEQ IDNO:84, there are employed a DNA containing the base sequence representedby SEQ ID NO:85, etc.; for the DNA encoding bovine GPR7 containing theamino acid sequence represented by SEQ ID NO:86, there are employed aDNA containing the base sequence represented by SEQ ID NO:87, etc.; and,for the DNA encoding bovine GPR8 containing the amino acid sequencerepresented by SEQ ID NO:88, there are employed a DNA containing thebase sequence represented by SEQ ID NO:89, etc.

[0496] The DNA encoding the partial peptide of GPR7 may be any DNA, aslong as it contains a base sequence encoding the partial peptide of GPR7described above. The DNA may also be any of genomic DNA, genomic DNAlibrary, cDNA derived from the cells and tissues described above, cDNAlibrary derived from the cells and tissues described above and syntheticDNA.

[0497] The DNA encoding the partial peptide of human GPR7 is, forexample, a DNA having a partial base sequence of the DNA having the basesequence represented by SEQ ID NO:50, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:50 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of human GPR7.

[0498] The DNA hybridizable to the base sequence represented by SEQ IDNO:50 has the same significance as described above.

[0499] The DNA encoding the partial peptide of rat TGR26 is, forexample, a DNA having a partial base sequence of the DNA having the basesequence represented by SEQ ID NO:60, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:60 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of rat TGR26.

[0500] The DNA hybridizable to the base sequence represented by SEQ IDNO:60 has the same significance as described above.

[0501] The DNA encoding the partial peptide of human GPR8 may be anyDNA, as long as it contains a base sequence encoding the partial peptideof human GPR8 described above. The DNA may also be any of genomic DNA,genomic DNA library, cDNA derived from the cells and tissues describedabove, cDNA library derived from the cells and tissues described aboveand synthetic DNA.

[0502] The DNA encoding the partial peptide of human GPR8 is, forexample, a DNA having a partial base sequence of the DNA having the basesequence represented by SEQ ID NO:85, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:85 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of human GPR8.

[0503] The DNA hybridizable to the base sequence represented by SEQ IDNO:85 has the same significance as described above.

[0504] The DNA encoding the partial peptide of bovine GPR7 may be anyDNA, as long as it contains a base sequence encoding the partial peptideof bovine GPR7 described above. The DNA may also be any of genomic DNA,genomic DNA library, cDNA derived from the cells and tissues describedabove, cDNA library derived from the cells and tissues described aboveand synthetic DNA.

[0505] The DNA encoding the partial peptide of bovine GPR7 is, forexample, a DNA having a partial base sequence of the DNA having the basesequence represented by SEQ ID NO:87, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:87 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of bovine GPR7.

[0506] The DNA hybridizable to the base sequence represented by SEQ IDNO:87 has the same significance as described above.

[0507] The DNA encoding the partial peptide of bovine GPR8 may be anyDNA, as long as it contains a base sequence encoding the partial peptideof bovine GPR8 described above. The DNA may also be any of genomic DNA,genomic DNA library, cDNA derived from the cells and tissues describedabove, cDNA library derived from the cells and tissues described aboveand synthetic DNA.

[0508] The DNA encoding the partial peptide of bovine GPR8 is, forexample, a DNA having a partial base sequence of the DNA having the basesequence represented by SEQ ID NO:89, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:89 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of bovine GPR8.

[0509] The DNA hybridizable to the base sequence represented by SEQ IDNO:89 has the same significance as described above.

[0510] With respect to the hybridization under high stringentconditions, the same procedures as described-above apply.

[0511] The DNA encoding the peptide of the present invention or GPR7 maybe labeled by publicly known methods. Specific examples include thoselabeled with an isotope, those labeled with fluorescence (labeling with,e.g., fluorescein, etc.), those biotinated, those labeled with enzyme,etc.

[0512] For cloning of the DNA that fully encodes the peptide of thepresent invention or GPR7, the DNA may be either amplified by publiclyknown PCR using synthetic DNA primers containing a part of the basesequence of the peptide of the present invention or GPR7, or the DNAinserted into an appropriate vector can be selected by hybridizationwith a labeled DNA fragment or synthetic DNA that encodes a part orentire region of the peptide of the present invention or GPR7. Thehybridization can be carried out, for example, according to the methoddescribed in Molecular Cloning, 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989), etc. The hybridization may also be performedusing commercially available library in accordance with the protocoldescribed in the attached instructions.

[0513] Conversion of the base sequence of DNA can be made by publiclyknown methods such as the ODA-LA PCR method, the Gapped duplex method orthe Kunkel method, or modifications thereof, by using a publicly knownkit available as Mutan™-super Express Km (manufactured by TaKaRa ShuzoCo., Ltd., trademark), Mutan™-K (manufactured by TaKaRa Shuzo Co., Ltd.,trademark), etc.

[0514] The cloned peptide-encoding DNA can be used as it is, dependingupon purpose or, if desired, after digestion with a restriction enzymeor after addition of a linker thereto. The DNA may contain ATG as atranslation initiation codon at the 5′ end thereof and TAA, TGA or TAGas a translation termination codon at the 3′ end thereof. Thesetranslation initiation and termination codons may also be added by usingan appropriate synthetic DNA adapter.

[0515] The expression vector of the peptide of the present invention orGPR7 can be manufactured, for example, by (a) excising the desired DNAfragment from the DNA encoding the peptide of the present invention orGPR7, (b) and then ligating the DNA fragment with an appropriateexpression vector downstream a promoter in the vector.

[0516] Examples of the vector include plasmids derived form E. coli(e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillussubtilis (e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g.,pSH19, pSH15), bacteriophages such as λ phage, etc., animal viruses suchas retrovirus, vaccinia virus, baculovirus, etc. as well as pA1-11,pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo, etc.

[0517] The promoter used in the present invention may be any promoter ifit matches well with a host to be used for gene expression. In the caseof using animal cells as the host, examples of the promoter include SRαpromoter, SV40 promoter, HIV•LTR promoter, CMV promoter, HSV-TKpromoter, etc.

[0518] Among them, CMV (cytomegalovirus) promoter or SRα promoter ispreferably used. Where the host is bacteria of the genus Escherichia,preferred examples of the promoter include trp promoter, lac promoter,recA promoter, λPL promoter, lpp promoter, T7 promoter, etc. In the caseof using bacteria of the genus Bacillus as the host, preferred exampleof the promoter are SPO1 promoter, SPO2 promoter and penP promoter. Whenyeast is used as the host, preferred examples of the promoter are PHO5promoter, PGK promoter, GAP promoter and ADH promoter. When insect cellsare used as the host, preferred examples of the promoter includepolyhedrin promoter, P10 promoter, etc.

[0519] In addition to the foregoing examples, the expression vector mayfurther optionally contain an enhancer, a splicing signal, a poly Aaddition signal, a selection marker, SV40 replication origin(hereinafter sometimes abbreviated as SV40ori) etc. Examples of theselection marker include dihydrofolate reductase (hereinafter sometimesabbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillinresistant gene (hereinafter sometimes abbreviated as Amp^(r)), neomycinresistant gene (hereinafter sometimes abbreviated as Neo^(r), G418resistance), etc. In particular, when dhfr gene is employed as theselection marker using dhfr gene-deficient Chinese hamster cells,selection can also be made on thymidine free media.

[0520] If necessary, a signal sequence that matches with a host is addedto the N-terminus of the peptide of the present invention. Examples ofthe signal sequence that can be used are Pho A signal sequence, OmpAsignal sequence, etc. in the case of using bacteria of the genusEscherichia as the host; α-amylase signal sequence, subtilisin signalsequence, etc. in the case of using bacteria of the genus Bacillus asthe host; MFα signal sequence, SUC2 signal sequence, etc. in the case ofusing yeast as the host; and insulin signal sequence, α-interferonsignal sequence, antibody molecule signal sequence, etc. in the case ofusing animal cells as the host, respectively.

[0521] Using the vector comprising the DNA encoding the peptide of thepresent invention thus constructed, transformants can be manufactured.

[0522] Examples of the host, which may be employed, are bacteriabelonging to the genus Escherichia, bacteria belonging to the genusBacillus, yeast, insect cells, insects and animal cells, etc.

[0523] Specific examples of the bacteria belonging to the genusEscherichia include Escherichia coli K12 DH1 [Proc. Natl. Acad. Sci.U.S.A., 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)],JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal ofMolecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)],etc.

[0524] Examples of the bacteria belonging to the genus Bacillus includeBacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal ofBiochemistry, 95, 87 (1984)], etc.

[0525] Examples of yeast include Saccharomyces cereviseae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,Pichia pastoris KM71, etc.

[0526] Examples of insect cells include, for the virus AcNPV, Spodopterafrugiperda cell (Sf cell), MG1 cell derived from mid-intestine ofTrichoplusia ni, High Five™ cell derived from egg of Trichoplusia ni,cells derived from Mamestra brassicae, cells derived from Estigmenaacrea, etc.; and for the virus BmNPV, Bombyx mori N cell (BmN cell),etc. is used. Examples of the Sf cell which can be used are Sf9 cell(ATCC CRL1711), Sf21 cell (both cells are described in Vaughn, J. L. etal., In Vivo, 13, 213-217 (1977)), etc.

[0527] As the insect, for example, a larva of Bombyx mori, etc. can beused [Maeda et al., Nature, 315, 592 (1985)].

[0528] Examples of animal cells include monkey cell COS-7, Vero, Chinesehamster cell CHO (hereinafter referred to as CHO cell), dhfr genedeficient Chinese hamster cell CHO (hereinafter simply referred to asCHO (dhfr⁻) cell), mouse L cell, mouse AtT-20, mouse myeloma cell, ratGH 3, human FL cell, etc.

[0529] Bacteria belonging to the genus Escherichia can be transformed,for example, by the method described in Proc. Natl. Acad. Sci. U.S.A.,69, 2110 (1972), Gene, 17, 107 (1982), etc.

[0530] Bacteria belonging to the genus Bacillus can be transformed, forexample, by the method described in Molecular & General Genetics, 168,111 (1979), etc.

[0531] Yeast can be transformed, for example, by the method described inMethods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci.U.S.A., 75, 1929 (1978), etc.

[0532] Insect cells or insects can be transformed, for example,according to the method described in Bio/Technology, 6, 47-55(1988),etc.

[0533] Animal cells can be transformed, for example, according to themethod described in Saibo Kogaku (Cell Engineering), extra issue 8, ShinSaibo Kogaku Jikken Protocol (New Cell Engineering ExperimentalProtocol), 263-267 (1995), published by Shujunsha, or Virology, 52, 456(1973).

[0534] Thus, the transformant transformed with the expression vectorcontaining the DNA encoding the peptide can be obtained.

[0535] Where the host is bacteria belonging to the genus Escherichia orthe genus Bacillus, the transformant can be appropriately cultured in aliquid medium which contains materials required for growth of thetransformant such as carbon sources, nitrogen sources, inorganicmaterials, etc. Examples of the carbon sources include glucose, dextrin,soluble starch, sucrose, etc. Examples of the nitrogen sources includeinorganic or organic materials such as ammonium salts, nitrate salts,corn steep liquor, peptone, casein, meat extract, soybean cake, potatoextract, etc. Examples of the inorganic materials are calcium chloride,sodium dihydrogenphosphate, magnesium chloride, etc. In addition, yeast,vitamins, growth promoting factors etc. may also be added to the medium.Preferably, pH of the medium is adjusted to about 5 to about 8.

[0536] A preferred example of the medium for culturing the bacteriabelonging to the genus Escherichia is M9 medium supplemented withglucose and Casamino acids [Miller, Journal of Experiments in MolecularGenetics, 431-433, Cold Spring Harbor Laboratory, New York, 1972]. Ifnecessary and desired, a chemical such as 3β-indolylacrylic acid can beadded to the medium thereby to activate the promoter efficiently.

[0537] Where the bacteria belonging to the genus Escherichia are used asthe host, the transformant is usually cultivated at approximately 15 to43° C. for approximately 3 to 24 hours. If necessary, the culture may beaerated or agitated.

[0538] Where the bacteria belonging to the genus Bacillus are used asthe host, the transformant is cultivated generally at approximately 30to 40° C. for approximately 6 to 24 hours. If necessary, the culture canbe aerated or agitated.

[0539] Where yeast is used as the host, the transformant is cultivated,for example, in Butkholder's minimal medium [Bostian, K. L. et al.,Proc. Natl. Acad. Sci. U.S.A., 77, 4505 (1980)] or in SD mediumsupplemented with 0.5% Casamino acids [Bitter, G. A. et al., Proc. Natl.Acad. Sci. U.S.A., 81, 5330 (1984)]. Preferably, pH of the medium isadjusted to about 5 to about 8. In general, the transformant iscultivated at approximately 20° C. to 35° C. for approximately 24 to 72hours. If necessary, the culture can be aerated or agitated.

[0540] Where insect cells or insects are used as the host, thetransformant is cultivated in, for example, Grace's Insect Medium(Grace, T. C. C., Nature, 195, 788 (1962)) to which an appropriateadditive such as immobilized 10% bovine serum is added. Preferably, pHof the medium is adjusted to about 6.2 to about 6.4. Normally, thetransformant is cultivated at about 27° C. for about 3 days to about 5days and, if necessary, the culture can be aerated or agitated.

[0541] Where animal cells are employed as the host, the transformant iscultivated in, for example, MEM medium containing about 5% to about 20%fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8,396 (1959)], RPMI 1640 medium [The Journal of the American MedicalAssociation, 199, 519 (1967)], 199 medium [Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)], etc. Preferably, pH of themedium is adjusted to about 6 to about 8. The transformant is usuallycultivated at about 30° C. to about 40° C. for about 15 hours to about60 hours and, if necessary, the culture can be aerated or agitated.

[0542] As described above, the peptide of the present invention or GPR7can be produced in the inside, cell membrane or outside of thetransformant, etc.

[0543] The peptide of the present invention or GPR7 can be separated andpurified from the culture described above, e.g., by the followingprocedures.

[0544] When the peptide of the present invention or GPR7 is extractedfrom the culture or cells, the transformant or cell is collected, aftercultivation, by a publicly known method and suspended in an appropriatebuffer. The transformant or cell is then disrupted by publicly knownmethods such as ultrasonication, a treatment with lysozyme and/orfreeze-thaw cycling, followed by centrifugation, filtration, etc. Thus,the crude extract of the peptide of the present invention or GPR7 can beobtained. The buffer used for the procedures may contain a proteinmodifier such as urea or guanidine hydrochloride, or a surfactant suchas Triton X-100™, etc. When the peptide is secreted in the culturebroth, after completion of the cultivation the supernatant can beseparated from the transformant or cell to collect the supernatant by apublicly known method.

[0545] The peptide of the present invention or GPR7 contained in thesupernatant or in the extract thus obtained can be purified byappropriately combining the publicly known methods for separation andpurification. Such publicly known methods for separation andpurification include a method utilizing difference in solubility such assalting out, solvent precipitation, etc.; a method mainly utilizingdifference in molecular weight such as dialysis, ultrafiltration, gelfiltration, SDS-polyacrylamide gel electrophoresis, etc.; a methodutilizing difference in electric charge such as ion exchangechromatography, etc.; a method utilizing difference in specific affinitysuch as affinity chromatography, etc.; a method utilizing difference inhydrophobicity such as reversed phase high performance liquidchromatography, etc.; a method utilizing difference in isoelectric pointsuch as isoelectrofocusing electrophoresis; and the like.

[0546] When the peptide of the present invention or GPR7 thus obtainedis in a free form, it can be converted into the salt by publicly knownmethods or modifications thereof. On the other hand, when the peptide isobtained in the form of a salt, it can be converted into the free formor in the form of a different salt by publicly known methods ormodifications thereof.

[0547] The peptide of the present invention or GPR7 produced by therecombinant can be treated, prior to or after the purification, with anappropriate protein modifying enzyme so that the peptide can beappropriately modified to partially remove a peptide. Examples of theprotein-modifying enzyme include trypsin, chymotrypsin, arginylendopeptidase, protein kinase, glycosidase and the like.

[0548] Antibodies to the peptide of the present invention (hereinaftersometimes simply referred to as the antibody(ies) of the presentinvention) may be any of polyclonal antibodies and monoclonalantibodies, as long as they are capable of recognizing antibodies to thepeptide of the present invention.

[0549] The antibodies to the peptide of the present invention may bemanufactured by publicly known methods for manufacturing antibodies orantisera, using as antigens the peptide of the present invention.

[0550] [Production of Monoclonal Antibody]

[0551] (a) Production of Monoclonal Antibody-Producing Cells

[0552] The peptide of the present invention is administered towarm-blooded animals either solely or together with carriers or diluentsto the site where the production of antibody is possible by theadministration. In order to potentiate the antibody productivity uponthe administration, complete Freund's adjuvants or incomplete Freund'sadjuvants may be administered. The administration is usually carried outonce every two to six weeks and two to ten times in total. Examples ofthe applicable warm-blooded animals are monkeys, rabbits, dogs, guineapigs, rice, rats, sheep, goats and chickens, with the use of mice andrats being preferred.

[0553] In the preparation of monoclonal antibody-producing cells, awarm-blooded animal, e.g., mice, immunized with an antigen wherein theantibody titer is noted is selected, then spleen or lymph node iscollected after two to five days from the final immunization andantibody-producing cells contained therein are fused with myeloma cellsfrom homozoic or heterozoic animal to give monoclonal antibody-producinghybridomas. Measurement of the antibody titer in antisera may be carriedout, for example, by reacting a labeled peptide, which will be describedlater, with the antiserum followed by assaying the binding activity ofthe labeling agent bound to the antibody. The fusion may be carried out,for example, by the known method by Koehler and Milstein [Nature, 256,495 (1975)]. Examples of the fusion promoter are polyethylene glycol(PEG), Sendai virus, etc., of which PEG is preferably employed.

[0554] Examples of the myeloma cells are those collected fromwarm-blooded animals such as NS-1, P3U1, SP2/0, AP-1, etc. Inparticular, P3U1 is preferably employed. A preferred ratio of the countof the antibody-producing cells used (spleen cells) to the count ofmyeloma cells is within a range of approximately 1:1 to 20:1. When PEG(preferably, PEG 1000 to PEG 6000) is added in a concentration ofapproximately 10 to 80% followed by culturing at 20 to 40° C.,preferably at 30 to 37° C. for 1 to 10 minutes, an efficient cell fusioncan be carried out.

[0555] Various methods can be used for screening of a monoclonalantibody-producing hybridoma. Examples of such methods include a methodwhich comprises adding the supernatant of hybridoma to a solid phase(e.g., microplate) adsorbed with the peptide (protein) as an antigendirectly or together with a carrier, adding an anti-immunoglobulinantibody (where mouse cells are used for the cell fusion, anti-mouseimmunoglobulin antibody is used) labeled with a radioactive substance oran enzyme or Protein A and detecting the monoclonal antibody bound tothe solid phase, and a method which comprises adding the supernatant ofhybridoma to a solid phase adsorbed with an anti-immunoglobulin antibodyor Protein A, adding the peptide labeled with a radioactive substance oran enzyme and detecting the monoclonal antibody bound to the solidphase.

[0556] The monoclonal antibody can be selected according to publiclyknown methods or their modifications. In general, the selection can beeffected in a medium for animal cells supplemented with HAT(hypoxanthine, aminopterin and thymidine). Any selection and growthmedium can be employed as far as the hybridoma can grow there. Forexample, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20%fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.)containing 1 to 10% fetal bovine serum, a serum free medium forcultivation of a hybridoma (SFM-101, Nissui Seiyaku Co., Ltd.) and thelike can be used for the selection and growth medium. The cultivation iscarried out generally at 20 to 40° C., preferably at 37° C., for about 5days to about 3 weeks, preferably 1 to 2 weeks, normally in 5% CO₂. Theantibody titer of the culture supernatant of a hybridoma can bedetermined as in the assay for the antibody titer in antisera describedabove.

[0557] (b) Purification of Monoclonal Antibody

[0558] Separation and purification of a monoclonal antibody can becarried out by publicly known methods, such as separation andpurification of immunoglobulins [for example, salting-out, alcoholprecipitation, isoelectric point precipitation, electrophoresis,adsorption and desorption with ion exchangers (e.g., DEAE),ultracentrifugation, gel filtration, or a specific purification methodwhich comprises collecting only an antibody with an activated adsorbentsuch as an antigen-binding solid phase, Protein A or Protein G anddissociating the binding to obtain the antibody].

[0559] [Production of Polyclonal Antibody]

[0560] The polyclonal antibody of the present invention can bemanufactured by publicly known methods or modifications thereof. Forexample, a warm-blooded animal is immunized with an immunogen (peptideantigen) per se, or a complex of immunogen and a carrier protein isformed and a warm-blooded animal is immunized with the complex in amanner similar to the method described above for the manufacture ofmonoclonal antibodies. The product containing the antibody to thepeptide of the present invention is collected from the immunized animalfollowed by separation and purification of the antibody.

[0561] In the complex of immunogen and carrier protein used to immunizea warm-blooded animal, the type of carrier protein and the mixing ratioof carrier to hapten may be any type and in any ratio, as long as theantibody is efficiently produced to the hapten immunized by crosslinkingto the carrier. For example, bovine serum albumin, bovine thyroglobulin,hemocyanin or the like is coupled to hapten in a carrier-to-haptenweight ratio of approximately 0.1 to 20, preferably about 1 to about 5.

[0562] A variety of condensation agents can be used for the coupling ofcarrier to hapten. Glutaraldehyde, carbodiimide, maleimide activatedester and activated ester reagents containing thiol group ordithiopyridyl group are used for the coupling.

[0563] The condensation product is administered to warm-blooded animalseither solely or together with carriers or diluents to the site that canproduce the antibody by the administration. In order to potentiate theantibody productivity upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration is usually made once approximately every 2 to 6 weeks andapproximately 3 to 10 times in total.

[0564] The polyclonal antibody can be collected from the blood, ascites,etc., preferably from the blood of warm-blooded animal immunized by themethod described above.

[0565] The polyclonal antibody titer in antiserum can be assayed by thesame procedure as that for the determination of serum antibody titerdescribed above. The separation and purification of the polyclonalantibody can be carried out, following the method for the separation andpurification of immunoglobulins performed as in the separation andpurification of monoclonal antibodies described hereinabove.

[0566] Antisense DNAs (hereinafter these DNAs are sometimes merelyreferred to as the antisense DNA) having a complementary orsubstantially complementary base sequence to the DNA encoding thepeptide of the present invention (hereinafter these DNAs are sometimesmerely referred to as the DNA of the present invention) can be anyantisense DNA, so long as they possess a base sequence complementary orsubstantially complementary to that of the DNA of the present inventionand capable of suppressing expression of the DNA.

[0567] The base sequence substantially complementary to the DNA of thepresent invention may, for example, be a base sequence having at leastabout 70% homology, preferably at least about 80% homology, morepreferably at least about 90% homology and most preferably at leastabout 95% homology, to the full-length base sequence or partial basesequence of the base sequence complementary to the DNA of the presentinvention (i.e., complementary strand to the DNA of the presentinvention). In the entire base sequence of the complementary strand tothe DNA of the present invention, an antisense DNA having at least about70% homology, preferably at least about 80% homology, more preferably atleast about 90% homology and most preferably at least about 95%homology, to the complementary strand of the base sequence which encodesthe N-terminal region of the peptide of the present invention (e.g., thebase sequence around the initiation codon). These antisense DNAs can besynthesized using a publicly known DNA synthesizer, etc.

[0568] Hereinafter, the utilities of (1) the peptide of the presentinvention; (2) the DNA of the present invention, (3) the antibody of thepresent invention, and (4) the antisense DNA are explained.

[0569] (1) Therapeutic/Preventive Agent for Diseases with which thePeptide of the Present Invention is Associated

[0570] As shown in EXAMPLE 6 later described, the peptide of the presentinvention has the cell stimulating activity on GPR7-expressed cells(e.g., the activity that promotes arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, change in cell membrane potential, phosphorylation ofintracellular proteins, activation of c-fos, pH reduction, GTPγ Sbinding activity, etc.), and is an endogenous ligand to GPR7. Moreover,the peptide of the present invention has an appetite (eating)stimulating activity, as shown in EXAMPLE 14 later described. Besides,the peptide of the present invention is considered to act as aneuromodulator or neuroendocrine substance or to be associated withmemory, learning or stress control.

[0571] Therefore, when the peptide of the present invention or the DNAof the present invention involves any abnormality or deficiency, or whenGPR7 or the DNA encoding GPR7 involves any abnormality or deficiency, itis highly likely to cause various diseases, including anorexia,hypertension, autoimmune disease, heart failure, cataract, glaucoma,acute bacterial meningitis, acute myocardial infarction, acutepancreatitis, acute viral encephalitis, adult respiratory distresssyndrome, alcoholic hepatitis, Alzheimer's disease, asthma,arteriosclerosis, atopic dermatitis, bacterial pneumonia, bladdercancer, fracture, breast cancer, bulimia, polyphagia, burn healing,uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, nbn-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative diseases (especially anorexia, etc.), or the like.

[0572] Therefore, the peptide of the present invention and the DNA ofthe present invention can be used as pharmaceuticals (in particular,appetite (eating) stimulants, etc.) for the treatment/prevention ofvarious diseases as described above (especially anorexia, etc.).

[0573] When a patient has a reduced level of, or deficient in thepeptide of the present invention in his or her body, the peptide of thepresent invention and the DNA of the present invention can provide therole of the peptide of the present invention sufficiently or properlyfor the patient, (a) by administering the DNA of the present inventionto the patient to express the peptide of the present invention in thebody, (b) by inserting the DNA of the present invention into a cell,expressing the peptide of the present invention and then transplantingthe cell to the patient, or (c) by administering the peptide of thepresent invention to the patient, or the like.

[0574] When the DNA of the present invention is used as thepreventive/therapeutic agents described above, the DNA is administereddirectly to human or other warm-blooded animal; alternatively, the DNAis inserted into an appropriate vector such as retrovirus vector,adenovirus vector, adenovirus-associated virus vector, etc. and thenadministered to human or other warm-blooded animal in a conventionalmanner. The DNA of the present invention may also be administered as anintact DNA, or prepared into pharmaceutical preparations together with aphysiologically acceptable carrier such as an adjuvant to assist itsuptake and administered by gene gun or through a catheter such as acatheter with a hydrogel.

[0575] Where the peptide of the present invention is used as theaforesaid therapeutic/preventive agents, the peptide is advantageouslyused on a purity level of at least 90%, preferably at least 95%, morepreferably at least 98% and most preferably at least 99%.

[0576] The peptide of the present invention can be used orally, forexample, in the form of tablets which may be sugar coated if necessary,capsules, elixirs, microcapsules etc., or parenterally in the form ofinjectable preparations such as a sterile solution and a suspension inwater or with other pharmaceutically acceptable liquid. Thesepreparations can be manufactured by mixing the peptide of the presentinvention with a physiologically acceptable known carrier, a flavoringagent, an excipient, a vehicle, an antiseptic agent, a stabilizer, abinder, etc. in a unit dosage form required in a generally acceptedmanner that is applied to making pharmaceutical preparations. The activeingredient in the preparation is controlled in such a dose that anappropriate dose is obtained within the specified range given.

[0577] Additives miscible with tablets, capsules, etc. include a bindersuch as gelatin, corn starch, tragacanth and gum arabic, an excipientsuch as crystalline cellulose, a swelling agent such as corn starch,gelatin, alginic acid, etc., a lubricant such as magnesium stearate, asweetening agent such as sucrose, lactose and saccharin, and a flavoringagent such as peppermint, akamono oil or cherry, etc. When the unitdosage is in the form of capsules, liquid carriers such as oils and fatsmay further be used together with the additives described above. Asterile composition for injection may be formulated according to aconventional manner used to make pharmaceutical compositions, e.g., bydissolving or suspending the active ingredients in a vehicle such aswater for injection with a naturally occurring vegetable oil such assesame oil and coconut oil, etc. to prepare the pharmaceuticalcomposition.

[0578] Examples of an aqueous medium for injection include physiologicalsaline and an isotonic solution containing glucose and other auxiliaryagents (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) and may beused in combination with an appropriate dissolution aid such as analcohol (e.g., ethanol or the like), a polyalcohol (e.g., propyleneglycol and polyethylene glycol), a nonionic surfactant (e.g.,polysorbate 80™ and HCO-50), etc. Examples of the oily medium includesesame oil and soybean oil, which may also be used in combination with adissolution aid such as benzyl benzoate and benzyl alcohol. Theprophylactic/therapeutic agent described above may further be formulatedwith a buffer (e.g., phosphate buffer, sodium acetate buffer, etc.), asoothing agent (e.g., benzalkonium chloride, procaine hydrochloride,etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol,etc.), a preservative (e.g., benzyl alcohol, phenol, etc.), anantioxidant, etc. The thus-prepared liquid for injection is normallyfilled in an appropriate ampoule.

[0579] The vector in which the DNA of the present invention is insertedmay also be prepared into pharmaceutical preparations in a mannersimilar to the procedures above. Such preparations are generally usedparenterally.

[0580] Since the thus obtained pharmaceutical preparation is safe andlow toxic, the preparation can be administered to human or otherwarm-blooded animals (e.g., rats, mice, guinea pigs, rabbits, chicken,sheep, swine, bovine, horses, cats, dogs, monkeys, etc.).

[0581] The dose of the peptide of the present invention varies dependingon target disease, subject to be administered, route for administration,etc.; in oral administration, e.g., for the treatment of anorexia, thedose is normally about 0.1 mg to about 100 mg, preferably about 1.0 toabout 50 mg, and more preferably about 1.0 to about 20 mg per day foradult (as 60 kg body weight). In parenteral administration, the singledose varies depending on subject to be administered, target disease,etc. but it is advantageous, e.g., for the treatment of anorexia toadminister the active ingredient intravenously at a daily dose of about0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and morepreferably about 0.1 to about 10 mg for adult (as 60 kg body weight).For other animal species, the corresponding dose as converted per 60 kgbody weight can be administered.

[0582] (2) Screening of Drug Candidate Compounds for Diseases

[0583] (2-1) Screening Method A

[0584] Since the peptide of the present invention has the function toact as the ligand to GPR7, the compounds or salts thereof that promotethe function of the peptide of the present invention can be used asdrugs for the treatment/prevention of diseases such as anorexia,hypertension, autoimmune disease, heart failure, cataract, glaucoma,acute bacterial meningitis, acute myocardial infarction, acutepancreatitis, acute viral encephalitis, adult respiratory distresssyndrome, alcoholic hepatitis, Alzheimer's disease, asthma,arteriosclerosis, atopic dermatitis, bacterial pneumonia, bladdercancer, fracture, breast cancer, bulimia, polyphagia, burn healing,uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially anorexia, etc.), or the like.

[0585] On the other hand, the compounds or salts thereof that inhibitthe function of the peptide of the present invention are useful as safeand low-toxic drugs for the prevention/treatment of, e.g., obesity(e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, etc.; as safe and low-toxic drugs for thetreatment/prevention (prolactin production suppressing agents) forpituitary tumor, diencephalon tumor, menstrual disorder, autoimmunedisease, prolactinoma, sterility, impotence, amenorrhea, lactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc., preferably, as safe and low-toxic drugsfor the prevention/treatment of obesity, hyperphagia, etc.

[0586] By using the peptide of the present invention, or by constructingthe expression system of the recombinant peptide of the presentinvention and using the receptor-binding assay system via the expressionsystem, compounds that alter the binding property between the peptide ofthe present invention and GPR7 (compounds that promote or inhibit theactivities of the peptide of the present invention) (e.g., peptide,protein, a non-peptide compound, a synthetic compound, fermentationproduct, etc.), or salts thereof, can be screened. Such compoundsinclude compounds (i.e., GPR7 agonists) that have the cell-stimulatingactivity of the peptide of the present invention (e.g., the activitythat promotes arachidonic acid release, acetylcholine release,intracellular Ca²⁺ release, intracellular cAMP production, intracellularcGMP production, inositol phosphate production, change in cell membranepotential, phosphorylation of intracellular proteins, activation ofc-fos, pH reduction, GTPγ S binding activity, etc.) mediated by GPR7;compounds having no such cell-stimulating activity (i.e., GPR7antagonists); and the like. The term “alters the binding property to theligand” is used to include both cases where binding to the ligand isinhibited and binding to the ligand is promoted.

[0587] Thus, the present invention provides:

[0588] a method of screening a compound or its salt that promotes orinhibits the activity of the peptide of the present invention, whichcomprises using the peptide of the present invention, more specifically:

[0589] a method of screening a compound that alters the binding propertybetween the peptide of the present invention and GPR7 (a compound thatpromotes or inhibits the activity of the peptide of the presentinvention) or its salt, which comprises comparing (i) the case whereinthe peptide of the present invention is brought in contact with GPR7 orits partial peptide (hereinafter they are sometimes merely referred toas GPR7) and (ii) the case wherein the peptide of the present inventionand a test compound are brought in contact with GPR7.

[0590] According to the screening method of the present invention, themethod comprises assaying, for example, the binding amount of the ligandto GPR7, the cell-stimulating activity, etc. (i) in the case wherein thepeptide of the present invention is brought in contact with GPR7 and(ii) in the case wherein the peptide of the present invention and a testcompound are brought in contact with GPR7, and comparing (i) and (ii).

[0591] Specifically, the screening method of the present inventionincludes:

[0592] (1) a method of screening a compound that alters the bindingproperty between the peptide of the present invention and GPR7 (acompound that promotes or inhibits the activity of the peptide of thepresent invention) or its salt, which comprises assaying the bindingamount of a labeled form of the peptide of the present invention toGPR7, (i) in the case wherein a labeled form of the peptide of thepresent invention is brought in contact with GPR7 and (ii) in the casewherein a labeled form of the peptide of the present invention and atest compound are brought in contact with GPR7, and comparing (i) and(ii);

[0593] (2) a method of screening a compound that alters the bindingproperty between the peptide of the present invention and GPR7 (acompound that promotes or inhibits the activity of the peptide of thepresent invention) or its salt, which comprises assaying the bindingamount of a labeled form of the peptide of the present invention to acell containing GPR7 or its cell membrane, (i) in the case wherein alabeled form of the peptide of the present invention is brought incontact with the cell containing GPR7 or its cell membrane and (ii) inthe case wherein a labeled form of the peptide of the present inventionand a test compound are brought in contact with the cell containing GPR7or its cell membrane, and comparing (i) and (ii);

[0594] (3) a method of screening a compound that alters the bindingproperty between the peptide of the present invention and GPR7 (acompound that promotes or inhibits the activity of the peptide of thepresent invention) or its salt, which comprises assaying the bindingamount of a labeled form of the peptide of the present invention toGPR7, (i) in the case wherein a labeled form of the peptide of thepresent invention is brought in contact with GPR7 expressed on a cellmembrane by culturing a transformant containing a DNA encoding GPR7 and(ii) in the case wherein a labeled form of the peptide of the presentinvention and a test compound are brought in contact with GPR7 expressedon a cell membrane by culturing a transformant containing a DNA encodingGPR7, and comparing (i) and (ii);

[0595] (4) a method of screening a compound that alters the bindingproperty between the peptide of the present invention and GPR7 (acompound that promotes or inhibits the activity of the peptide of thepresent invention) or its salt, which comprises assaying thecell-stimulating activity mediated by GPR7 (e.g., the activity thatpromotes or suppresses arachidonic acid release, acetylcholine release,intracellular Ca²⁺ release, intracellular cAMP production, intracellularcGMP production, inositol phosphate production, change in cell membranepotential, phosphorylation of intracellular proteins, activation ofc-fos, pH reduction, GTPγ S binding activity, etc.), when a compoundthat activates GPR7 (e.g., the peptide of the present invention) isbrought in contact with a cell containing GPR7 and when the compoundthat activates GPR7 and a test compound are brought in contact with acell containing GPR7, and comparing the activity; and,

[0596] (5) a method of screening a compound that alters the bindingproperty between the peptide of the present invention and GPR7 (acompound that promotes or inhibits the activity of the peptide of thepresent invention) or its salt, which comprises assaying thecell-stimulating activity mediated by GPR7 (e.g., the activity thatpromotes or suppresses arachidonic acid release, acetylcholine release,intracellular Ca²⁺ release, intracellular cAMP production, intracellularcGMP production, inositol phosphate production, change in cell membranepotential, phosphorylation of intracellular proteins, activation ofc-fos, pH reduction, GTPγ S binding activity, etc.), when a compoundthat activates GPR7 (e.g., the peptide of the present invention, etc.)is brought in contact with GPR7 expressed on a cell membrane byculturing a transformant containing a DNA encoding GPR7 and when thecompound that activates GPR7 and a test compound are brought in contactwith GPR7 expressed on a cell membrane by culturing a transformantcontaining a DNA encoding GPR7, and comparing the activity; etc.

[0597] The screening method of the present invention will be describedbelow more specifically.

[0598] First, the GPR7, which is used for the screening method of thepresent invention, may be any protein, so long as it recognizes thepeptide of the present invention as a ligand, and membrane fractionsfrom human or other warm-blooded animal organs are preferably employed.However, it is very difficult to obtain human-derived organs especially,and the GPR7, etc. expressed abundantly by use of recombinants aresuitable for use in the screening. GPR7 may be manufactured by themethods described above.

[0599] Where the cell containing GPR7 or its cell membrane fraction isused in the screening method of the present invention, the procedureslater described may apply.

[0600] When the cell containing GPR7 is used, the cell may be fixed withglutaraldehyde, formalin, etc. The fixation may be carried out by apublicly known method.

[0601] The cell containing GPR7 refers to a host cell expressing GPR7.Examples of such a host cell include Escherichia coli, Bacillussubtilis, yeast, insect cells, animal cells, etc. Host cells in whichGPR7 is expressed may be prepared in a manner similar to theabove-stated method for manufacturing transformants transformed byexpression vectors containing the peptide of the present invention.

[0602] The membrane fraction refers to a fraction that abundantlycontains cell membranes prepared by publicly known methods afterdisrupting cells. Examples of the cell disruption include cell squashingusing a Potter-Elvehjem homogenizer, disruption using a Waring blenderor Polytron (manufactured by Kinematica Inc.), disruption byultrasonication, disruption by cell spraying via a thin nozzle underincreasing pressure using a French press, etc., and the like. Cellmembranes are fractionated mainly by fractionation using a centrifugalforce such as for fractionation centrifugation, density gradientcentrifugation, etc. For example, cell disruption fluid is centrifugedat a low rate (500 rpm to 3,000 rpm) for a short period of time(normally about 1 minute to about 10 minutes), the resulting supernatantis then centrifuged at a higher rate (15,000 rpm to 30,000 rpm) normallyfor 30 minutes to 2 hours. The precipitate thus obtained is used as themembrane fraction. The membrane fraction is rich in GPR7 expressed andmembrane components such as cell-derived phospholipids, membraneproteins, or the like.

[0603] The amount of GPR7 contained in the cells containing GPR7 or inthe membrane fraction is preferably 10³ to 10⁸ molecules per cell, morepreferably 10⁵ to 10⁷ molecules per cell. As the amount of expressionincreases, the ligand binding activity per unit of membrane fraction(specific activity) increases so that not only the highly sensitivescreening system can be constructed but also large quantities of samplescan be assayed with the same lot.

[0604] To perform the methods (1) through (3) for screening the compoundthat alters the binding property between the peptide of the presentinvention and GPR7 (the compound that promotes or inhibits the activityof the peptide of the present invention), an appropriate GPR7 fractionand a labeled form of the peptide of the present invention, etc. arerequired. The GPR7 fraction is preferably a fraction of a naturallyoccurring form of GPR7 or a fraction of a recombinant type of GPR7having an equivalent activity. Herein, the term equivalent activity isintended to mean a ligand binding activity, etc. that is equivalent tothe activity possessed by naturally occurring GPR7. As the labeledligand, there may be used a labeled ligand, a labeled ligand analogcompound, etc. For example, there may be used ligands that are labeledwith [³H], [¹²⁵I], [¹⁴C], [³⁵S], etc. Of these, [¹²⁵I]-labeled ligand ispreferred.

[0605] Specifically, the compound that alters the binding propertybetween the peptide of the present invention and GPR7 is screened by thefollowing procedures. First, a receptor preparation is prepared bysuspending cells containing GPR7 or the membrane fraction thereof in abuffer appropriate for use in the screening method. Any buffer can beused so long as it does not interfere the ligand-receptor binding,including a phosphate buffer or a Tris-HCl buffer, having pH of 4 to 10(preferably pH of 6 to 8), etc. For the purpose of minimizingnon-specific binding, a surfactant such as CHAPS, Tween-80™ (Kao-AtlasInc.), digitonin, deoxycholate, etc., may optionally be added to thebuffer. Further for the purpose of suppressing the degradation of GPR7or the peptide of the present invention with a protease, a proteaseinhibitor such as PMSF, leupeptin, E-64 (manufactured by PeptideInstitute, Inc.), pepstatin, etc. may also be added. A given amount(5,000 cpr to 500,000 cpm) of the labeled peptide of the presentinvention is added to 0.01 ml to 10 ml of the receptor solution, inwhich 10⁻¹⁰ M to 10⁻⁷ M of a test compound is co-present. To determinethe amount of non-specific binding (NSB), a reaction tube charged withan unlabeled form of the peptide of the present invention in a largeexcess is also provided. The reaction is carried out at approximately 0°C. to 50° C., preferably 4° C. to 37° C. for 20 minutes to 24 hours,preferably 30 minutes to 3 hours. After completion of the reaction, thereaction mixture is filtrated through glass fiber filter paper, etc. andwashed with an appropriate volume of the same buffer. The residualradioactivity on the glass fiber filter paper is then measured by meansof a liquid scintillation counter or γ-counter. When nonspecific binding(NSB) is subtracted from the count (B₀) where any antagonizing substanceis absent and the resulting count (B₀ minus NSB) is made 100%, the testcompound showing the specific binding amount (B minus NSB) of, e.g., 50%or less may be selected as a candidate compound.

[0606] The method (4) or (5) described above for screening the compoundthat alters the binding property between the peptide of the presentinvention and GPR7 (the compound that promotes or inhibits the activityof the peptide of the present invention) can be carried out as follows.For example, the cell stimulating activity mediated by GPR7 (e.g., theactivity that promotes or suppresses arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, change in cell membrane potential, phosphorylation ofintracellular proteins, activation of c-fos, pH reduction, GTPγ Sbinding activity, etc.) may be determined by a publicly known method, orusing an assay kit commercially available. Specifically, the cellscontaining GPR7 are first cultured on a multiwell plate, etc. Prior toscreening, the medium is replaced with fresh medium or with anappropriate non-cytotoxic buffer, followed by incubation for a givenperiod of time in the presence of a test compound, etc. Subsequently,the cells are extracted or the supernatant is recovered and theresulting product is quantified by appropriate procedures. Where it isdifficult to detect the production of the cell-stimulating activityindicator (e.g., arachidonic acid, etc.) due to a degrading enzymecontained in the cells, an inhibitor against such as a degrading enzymemay be added prior to the assay. For detecting the activity such as thecAMP production suppression, the baseline production in the cells isincreased by forskolin or the like and the suppressing effect on theincreased baseline production can be detected.

[0607] For screening through the assay of the cell stimulating activity,appropriate cells, in which GPR7 is expressed, are required. Preferredcells, in which GPR7 is expressed, are the aforesaid cell line in whichGPR7 is expressed, etc.

[0608] Examples of the test compounds include peptides, proteins,non-peptide compounds, synthetic compounds, fermentation products, cellextracts, plant extracts, animal tissue extracts, etc.

[0609] The kit for screening the compound or a salt thereof that altersthe binding property between the peptide of the present invention (thecompound that promotes or inhibits the activity of the peptide of thepresent invention) and GPR7 comprises GPR7 or its salt, a partialpeptide of GPR7 or its salt, cells containing GPR7 or a membranefraction of the cells containing GPR7, and the peptide of the presentinvention.

[0610] Examples of the screening kit of the present invention are givenbelow:

[0611] 1. Reagent for Screening

[0612] (1) Assay Buffer and Wash Buffer

[0613] Hanks' Balanced Salt Solution (manufactured by Gibco Co.)supplemented with 0.05% bovine serum albumin (Sigma Co.).

[0614] The solution is sterilized by filtration through a 0.45 μm filterand stored at 4° C. Alternatively, the solution may be prepared at use.

[0615] (2) GPR7 Preparation

[0616] CHO cells on which GPR7 has been expressed are subcultured in a12-well plate at the rate of 5×10⁵ cells/well and then cultured at 37°C. under 5% CO₂ and 95% air for 2 days.

[0617] (3) Labeled Ligand

[0618] The peptide of the present invention labeled with commerciallyavailable [³H], [¹²⁵I], [¹⁴C], [³⁵S], etc. is dissolved in a suitablesolvent or buffer. The solution is stored at 4° C. or −20° C., which isdiluted to 1 μM with an assay buffer at use.

[0619] (4) Standard Ligand Solution

[0620] The peptide of the present invention is dissolved in PBSsupplemented with 0.1% bovine serum albumin (manufactured by Sigma,Inc.) in a concentration of 1 mM, and the solution is stored at −20° C.

[0621] 2. Assay Method

[0622] (1) Cells are cultured in a 12-well tissue culture plate toexpress GPR7. After washing the cells twice with 1 ml of the assaybuffer, 490 μl of the assay buffer is added to each well.

[0623] (2) After 5 μl of a test compound solution of 10⁻³ to 10⁻¹⁰ M isadded, 5 μl of a labeled form of the peptide of the present invention isadded to the system followed by reacting at room temperature for anhour. To determine the amount of the non-specific binding, the peptideof the present invention of 10⁻³ M is added in an amount of 5 μl,instead of the test compound.

[0624] (3) The reaction mixture is removed and washed 3 times with 1 mleach of the wash buffer. The labeled peptide of the present inventionbound to the cells is dissolved in 0.2N NaOH-1% SDS and mixed with 4 mlof a liquid scintillator A (manufactured by Wako Pure ChemicalIndustries, Ltd.).

[0625] (4) Radioactivity is measured using a liquid scintillationcounter (manufactured by Beckmann) and PMB (percent of the maximumbinding) is calculated in accordance with the following equation 1:

PMB[(B−NSB)/(B ₀ −NSB)]×100

[0626] wherein:

[0627] PMB: percent of the maximum binding

[0628] B: value when a sample is added

[0629] NSB: non-specific binding

[0630] B₀: maximum binding

[0631] The compound or its salt, which can be obtained by the screeningmethod or the screening kit of the present invention, is the compoundthat alters the binding property between the peptide of the presentinvention and GPR7 (the compound that promotes or inhibits the activityof the peptide of the present invention). Specifically, these compoundsare compounds or salts thereof that exhibit the cell stimulatingactivity mediated by GPR7 (i.e., GPR7 agonist), or compounds that haveno such cell stimulating activity (i.e., GPR7 antagonist). Examples ofsuch compounds-include peptides, proteins, non-peptide compounds,synthetic compounds and fermentation products. These compounds may beeither novel or publicly known compounds.

[0632] In order to evaluate whether the compound is either the GPR7agonist or antagonist described above, it is determined by (i) or (ii)below.

[0633] (i) According to the screening methods (1) to (3), binding assayis carried out to obtain the compound that alters the binding propertybetween the peptide of the present invention and GPR7 (especially, thecompound that inhibits the binding). It is then determined if thecompound has the above cell-stimulating activity mediated by GPR7. Thecompound or its salt having the cell-stimulating activity is the GPR7agonist, whereas the compound or its salt having no such an activity isthe GPR7 antagonist.

[0634] (ii) (a) A test compound is brought in contact with a cellcontaining GPR7, whereby the aforesaid cell-stimulating activitymediated by GPR7 is assayed. The compound having the cell-stimulatingactivity or its salt is the GPR7 agonist.

[0635] (b) The cell-stimulating activity mediated by GPR7 is assayed inthe case where a compound that activates GPR7 (e.g., the peptide of thepresent invention or GPR7 agonist, etc.) is brought in contact withcells containing GPR7 and in the case where the compound that activatesGPR7 and a test compound are brought in contact with cells containingGPR7, and comparison is made therebetween. The compound or its salt thatcan reduce the cell-stimulating activity induced by the compound thatactivates GPR7 is the GPR7 antagonist.

[0636] The GPR7 agonists exhibit similar physiological activity of thepeptide of the present invention on GPR7, and are thus safe andlow-toxic drugs (e.g., preventive/therapeutic drugs for anorexia,appetite (eating) stimulants, preventive/therapeutic drugs for pituitaryhormone secretion disorders [e.g., prolactin secretion disorders (e.g.,hypoovarianism, spermatic underdevelopment, menopausal symptoms,hypothyroidism, etc.)].

[0637] On the contrary, the GPR7 antagonist can suppress thephysiological activity that the peptide of the present invention has onGPR7, and are thus useful as safe and low-toxic drugs for theprevention/treatment of, e.g., obesity (e.g., malignant mastocytosis,exogenous obesity, hyperinsulinar obesity, hyperplasmic obesity,hypophyseal adiposity, hypoplasmic obesity, hypothyroid obesity,hypothalamic obesity, symptomatic obesity, infantile obesity, upper bodyobesity, alimentary obesity, hypogonadal obesity, systemic mastocytosis,simple obesity, central obesity, etc.), hyperphagia, etc.; as safe andlow-toxic drugs for the treatment/prevention (prolactin productionsuppressing agents) for pituitary tumor, diencephalon tumor, menstrualdisorder, autoimmune disease, prolactinoma, sterility, impotence,amenorrhea, lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-delCastillo syndrome, Forbes-Albright syndrome, lymphoma or Sheehan'ssyndrome, spermatogenesis disorder, etc.; preferably as safe andlow-toxic preventive/therapeutic agents for obesity, hyperphagia, etc.

[0638] The compound or its salt, which can be obtained by using thescreening method A or the screening kit of the present invention, isselected from, e.g., peptides, proteins, non-peptide compounds,synthetic compounds, fermentation products, cell extracts, plantextracts, animal tissue extracts, plasma, etc., and is the compound thatpromotes or inhibits the function of the peptide of the presentinvention.

[0639] As salts of the compound, those similar to the salts of thepeptide of the present invention described above may be used.

[0640] When the compound obtained by the screening method A or screeningkit of the present invention is used as the therapeutic/preventive agentdescribed above, the compound can be prepared into pharmaceuticalpreparations in a conventional manner. For example, the compound may beprepared in the form of tablets, capsules, elixir, microcapsule, asterile solution, a suspension, etc., as in the aforesaid drugscontaining the peptide of the present invention.

[0641] Since the thus obtained pharmaceutical preparation is safe andlow toxic, the preparation may be administered to human or otherwarm-blooded animals (e.g., mice, rats, rabbits, sheep, swine, bovine,horses, chicken, cats, dogs, monkeys, chimpanzees, etc.).

[0642] The dose of the compound or its salt varies depending on itsactivity, target disease, subject to be administered, route foradministration, etc.; where the GPR7 agonist is orally administered,e.g., for the treatment of anorexia, the dose of the compound isnormally about 0.1 to about 100 mg, preferably about 1.0 to about 50 mg,and more preferably about 1.0 to about 20 mg per day for adult (as 60 kgbody weight). In parenteral administration, a single dose of thecompound varies depending on subject to be administered, target disease,etc. When the GPR7 agonist is administered to adult (as 60 kg bodyweight) in the form of injection, e.g., for the treatment of anorexia,it is advantageous to administer the compound intravenously to adultgenerally at a daily dose of about 0.01 to about 30 mg, preferably about0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg. Forother animal species, the corresponding dose as converted per 60 kg bodyweight can be administered.

[0643] Also, when the GPR7 antagonist is orally administered to adult(per 60 kg body weight), e.g., for the treatment of obesity, a dailydose of the compound administered is generally approximately 0.1 to 100mg, preferably approximately 1.0 to 50 mg, and more preferablyapproximately 1.0 to 20 mg. In parenteral administration, a single doseof the compound varies depending on subject to be administered, targetdisease, etc. When the GPR7 antagonist is administered to adult (as 60kg body weight) in the form of injection, e.g., for the treatment ofobesity, it is advantageous to administer the compound intravenously toadult (per 60 kg body weight) generally at a daily dose of about 0.01 toabout 30 mg, preferably about 0.1 to about 20 mg, and more preferablyabout 0.1 to about 10 mg. For other animal species, the correspondingdose as converted per 60 kg body weight can be administered.

[0644] (2-2) Screening Method B

[0645] Next, the method of screening a compound that regulates theexpression level of GPR7 ligand is explained below.

[0646] The screening method B of the present invention is specifically(i) a method of screening a compound or its salt that increases ordecreases the expression level of GPR7 ligand, which comprises assayingthe expression level of GPR7 ligand or the amount of mRNA encoding GPR7ligand in the case that a cell or tissue capable of expressing GPR7ligand is cultured in the presence or absence of a test compound, andcomparing the expression level in each case.

[0647] As the cell or tissue capable of expressing GPR7 ligand, theremay be used a cell or tissue derived from human or other warm-bloodedanimals (e.g. guinea pigs, rats, mice, chicken, rabbits, swine, sheep,bovine, monkeys, etc.); any cell (e.g., nerve cells, endocrine cells,neuroendocrine cells, glial cells, β cells of pancreas, bone marrowcells, hepatocytes, splenocytes, mesangial cells, epidermic cells,epithelial cells, endothelial cells, fibroblasts, fibrocytes, myocytes,fat cells, immune cells (e.g., macrophages, T cells, B cells, naturalkiller cells, mast cells, neutrophils, basophils, eosinophils,monocytes, dendritic cells), megakaryocyte, synovial cells,chondrocytes, bone cells, osteoblasts, osteoclasts, mammary gland cellsor interstitial cells, the corresponding precursor cells, stem cells,cancer cells, etc., of these cells), or any tissue where such a cell ispresent, e.g., brain or any region of the brain (e.g., olfactory bulb,amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus,cerebral cortex, medulla oblongata, cerebellum), spinal cord,hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid,gall-bladder, bone marrow, adrenal gland, skin, muscle, lung,gastrointestinal tract (e.g., large intestine and small intestine),blood vessel, heart, thymus, spleen, submandibular gland, peripheralblood, prostate, testis, ovary, placenta, uterus, bone, cartilage,joint, skeletal muscle, etc., wherein established cell line or primaryculture system may also be used. Transformants transformed by arecombinant vector bearing a DNA encoding GPR7 ligand described abovemay also be used.

[0648] To cultivate the cells capable of expressing GPR7 ligand, themethod given for cultivating transformants above applies.

[0649] As the test compound, a DNA library may also be used, in additionto the test compounds described above.

[0650] The expression level of GPR7 ligand can be determined by publiclyknown methods such as immunochemical methods, etc., using an antibody,etc. Alternatively, mRNA encoding GPR7 ligand can be determined bypublicly known methods including northern hybridization, RT-PCR orTaqMan PCR.

[0651] Comparison of the expression level of mRNA can be made bypublicly known methods or a modification thereof, for example, accordingto the method described in Molecular Cloning, 2nd (J. Sambrook et al.,Cold Spring Harbor Lab. Press, 1989).

[0652] Specifically, the amount of mRNA encoding GPR7 ligand isdetermined by contacting RNA extracted from cells according to publiclyknown methods with the DNA encoding GPR7 ligand or a part thereof or theantisense polynucleotide of the present invention, and assaying theamount of mRNA bound to the DNA encoding GPR7 ligand or a part thereofor the antisense polynucleotide of the present invention. The amount ofmRNA bound to the DNA encoding GPR7 ligand or a part thereof or theantisense polynucleotide of the present invention can be readily assayedby labeling the DNA encoding GPR7 ligand or a part thereof or theantisense polynucleotide of the present invention with, e.g., aradioisotope, a dye, etc. Examples of the radioisotope are ³²P, ³H, etc.Examples of the dye used are fluorescent dyes such as fluorescein, FAM(Biosystems, Inc.), JOE (PE Biosystems, Inc.), TAMRA (PE Biosystems,Inc.), ROX (PE Biosystems, Inc.), Cy5 (Amersham), Cy3 (Amersham), etc.

[0653] The amount of mRNA can also be determined by converting RNAextracted from cells into cDNA by a reverse transcriptase, amplifyingthe cDNA by PCR using the DNA encoding GPR7 ligand or a part thereof orthe antisense polynucleotide of the present invention as a primer, andassaying the amount of cDNA amplified.

[0654] As described above, the test compound that increases the amountof mRNA encoding GPR7 ligand can be selected as a compound thatincreases the expression level of GPR7 ligand. Also, the test compoundthat decreases the amount of mRNA encoding GPR7 ligand can be selectedas a compound that decreases the expression level of GPR7 ligand.

[0655] The present invention further provides:

[0656] (ii) a method of screening a compound or its salt that promotesor inhibits a reporter activity, which comprises assaying the expressionlevel of GPR7 ligand or the amount of mRNA encoding GPR7 ligand in thecase that a cell or tissue capable of expressing GPR7 ligand is culturedin the presence or absence of a test compound, and comparing theexpression level in each case.

[0657] As the reporter gene, there may be employed, e.g., lacZ(β-galactosidase gene), chloramphenicol acetyltransferase (CAT),luciferase, growth factor, β-glcuronidase, alkaline phosphatase, greenfluorescent protein (GFP), β-lactamase, etc.

[0658] By determining the level of the reporter gene product (e.g.,mRNA, protein) Using publicly known methods, the test compound thatincreases the level of the reporter gene product can be selected as thecompound having the activity of regulating (especially promoting) thepromoter or enhancer activity of GPR7 ligand of the present invention,i.e., the compound having the activity of increasing the expressionlevel of GPR7 ligand. To the contrary, the test compound that decreasesthe level of the reporter gene product can be selected as the compoundhaving the activity of regulating (especially inhibiting) the promoteror enhancer activity of GPR7 ligand, i.e., the compound having theactivity of decreasing the expression level of GPR7 ligand.

[0659] As the test compounds, those described above are employed.

[0660] The transformants can be cultivated as in the transformantsdescribed above.

[0661] Construction of vectors for the reporter genes and assay can beperformed according to publicly known techniques (e.g., MolecularBiotechnology, 13, 29-43, 1999).

[0662] The compounds having the activity of increasing the expressionlevel of GPR7 are useful as safe and low-toxic drugs (e.g.,preventive/therapeutic agents for anorexia, appetite (eating)stimulants, preventive/therapeutic agents for pituitary hormonesecretion disorders [e.g., prolactin secretion disorders (e.g.,hypoovarianism, spermatic underdevelopment, menopausal symptoms,hypothyroidism, etc.)].

[0663] The compounds having the activity of decreasing the expressionlevel of GPR7 ligand are useful as safe and low-toxic drugs for theprevention/treatment of obesity (e.g., malignant mastocytosis, exogenousobesity, hyperinsulinar obesity, hyperplasmic obesity, hypophysealadiposity, hypoplasmic obesity, hypothyroid obesity, hypothalamicobesity, symptomatic obesity, infantile obesity, upper body obesity,alimentary obesity, hypogonadal obesity, systemic mastocytosis, simpleobesity, central obesity, etc.), hyperphagia, etc.; as safe andlow-toxic drugs for the prevention/treatment (prolactin productionsuppressing agents) for pituitary tumor, diencephalon tumor, menstrualdisorders, autoimmune disease, prolactinoma, sterility, impotence,amenorrhea, lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-delCastillo syndrome, Forbes-Albright syndrome, lymphoma or Sheehan'ssyndrome, spermatogenesis disorder, etc.; preferably, as safe andlow-toxic drugs for the prevention/treatment of obesity, hyperphagia,etc.

[0664] The compound or its salt, which can be obtained by using thescreening method B or the screening kit of the present invention, is acompound selected from, e.g., peptides, proteins, non-peptide compounds,synthetic compounds, fermentation products, cell extracts, plantextracts, animal tissue extracts, plasma, etc., and is the compound thatpromotes or inhibits the function of the peptide of the presentinvention.

[0665] For salts of the compound, those as described for the peptide ofthe present invention are employed.

[0666] When the compound obtained by the screening method B or screeningkit of the present invention is used as the therapeutic/preventive agentdescribed above, the compound can be prepared into pharmaceuticalpreparations in a conventional manner. For example, the compound may beprepared in the form of tablets, capsules, elixir, microcapsule, asterile solution, a suspension, etc., as in the aforesaid drugscontaining the peptide of the present invention.

[0667] Since the thus obtained pharmaceutical preparation is safe andlow toxic, the preparation may be administered to human or otherwarm-blooded animals (e.g., mice, rats, rabbits, sheep, swine, bovine,horses, chicken, cats, dogs, monkeys, chimpanzees, etc.).

[0668] The dose of the compound or its salt varies depending on itsactivity, target disease, subject to be administered, route foradministration, etc.; where the compound that increases the expressionlevel of GPR7 ligand is orally administered, e.g., for the treatment ofanorexia, the dose of the compound is normally about 0.1 to about 100mg, preferably about 1.0 to about 50 mg, and more preferably about 1.0to about 20 mg per day for adult (as 60 kg body weight). In parenteraladministration, a single dose of the compound varies depending onsubject to be administered, target disease, etc. When the compound thatincreases the expression level of GPR7 ligand is administered to adult(as 60 kg body weight) in the form of injection, e.g., for the treatmentof anorexia, it is advantageous to administer the compound intravenouslyto adult generally at a daily dose of about 0.01 to about 30 mg,preferably about 0.1 to about 20 mg, and more preferably about 0.1 toabout 10 mg. For other animal species, the corresponding dose asconverted per 60 kg body weight can be administered.

[0669] Also, when the compound that decreases the expression level ofGPR7 ligand is orally administered to adult (per 60 kg body weight),e.g., for the treatment of obesity, a daily dose of the compoundadministered is generally approximately 0.1 to 100 mg, preferablyapproximately 1.0 to 50 mg, and more preferably approximately 1.0 to. 20mg. In parenteral administration, a single dose of the compound variesdepending on subject to be administered, target disease, etc. When thecompound that decreases the expression level of GPR7 ligand isadministered to adult (as 60 kg body weight) in the form of injection,e.g., for the treatment of obesity, it is advantageous to administer thecompound intravenously to adult (per 60 kg body weight) generally at adaily dose of about 0.01 to about 30 mg, preferably about 0.1 to about20 mg, and more preferably about 0.1 to about 10 mg. For other animalspecies, the corresponding dose as converted per 60 kg body weight canbe administered.

[0670] (3) Quantification of the Peptide of the Present Invention

[0671] The antibody of the present invention is capable of specificallyrecognizing the peptide of the present invention, and can thus be usedfor quantification of the peptide of the present invention in a samplefluid, in particular, for quantification by sandwich immunoassay.

[0672] That is, the present invention provides:

[0673] (i) a method for quantification of the peptide of the presentinvention in a sample fluid, which comprises competitively reacting theantibody of the present invention with a sample fluid and a labeled formof the peptide of the present invention, and measuring a ratio of thelabeled peptide of the present invention bound to the antibody; and,

[0674] (ii) a method for quantification of the peptide of the presentinvention in a sample fluid, which comprises simultaneously orcontinuously reacting the sample fluid with the antibody of the presentinvention and a labeled form of another antibody of the presentinvention immobilized on an insoluble carrier, and measuring theactivity of the labeling agent on the immobilized carrier.

[0675] In the method of quantification (ii) described above, it ispreferred that one antibody is capable of recognizing the N-terminalregion of the peptide of the present invention, while another antibodyis capable of recognizing the C-terminal region of the peptide of thepresent invention.

[0676] The monoclonal antibody to the peptide of the present inventionmay be used to quantify the peptide of the present invention. Moreover,the peptide of the present invention may also be detected by means of atissue staining, etc. For these purposes, the antibody molecule per semay be used, or F(ab′)₂, Fab′ or Fab fractions of the antibody moleculemay be used as well.

[0677] The method of quantifying the peptide of the present inventionusing the antibody of the present invention is not particularly limited,and any method may be used so far as it relates to a method, in whichthe amount of an antibody, antigen or antibody-antigen complex can bedetected by a chemical or a physical means, depending on orcorresponding to the amount of antigen (e.g., the amount of the peptide)in a sample fluid to be assayed, and then calculated using a standardcurve prepared by a standard solution containing the known amount ofantigen. Advantageously used are, for example, nephrometry, competitivemethod, immunometric method and sandwich method; in terms of sensitivityand specificity, the sandwich method, which will be described later, isparticularly preferred.

[0678] Examples of labeling agents, which are employed for the assaymethod using the same, are radioisotopes, enzymes, fluorescentsubstances, luminescent substances, etc. Examples of radioisotopes are[¹²⁵I], [¹³¹I], [³H], [¹⁴C], etc. Preferred examples of enzymes arethose that are stable and have a high specific activity, which includeβ-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malatedehydrogenase, etc. Examples of fluorescent substances arefluorescamine, fluorescein isothiocyanate, etc. Examples of luminescentsubstances are luminol, a luminol derivative, luciferin, lucigenin, etc.Furthermore, a biotin-avidin system may be used as well for binding anantibody or antigen to a labeling agent.

[0679] In the immobilization of antigens or antibodies, physicaladsorption may be used. Alternatively, chemical binding that isconventionally used for immobilization of proteins, enzymes, etc. may beused as well. Examples of the carrier include insoluble polysaccharidessuch as agarose, dextran, cellulose, etc.; synthetic resins such aspolystyrene, polyacrylamide, silicone, etc.; or glass; and the like.

[0680] In the sandwich method, a sample fluid is reacted with animmobilized form of the monoclonal antibody of the present invention(primary reaction), then reacted with a labeled form of the monoclonalantibody of the present invention (secondary reaction) and the activityof the labeling agent on the insoluble carrier is assayed; thus, theamount of the peptide of the present invention in a sample fluid can bedetermined. The primary and secondary reactions may be carried out in areversed order, simultaneously or sequentially with intervals. The typeof the labeling agent and the method of immobilization may be the sameas those described hereinabove. In the immunoassay by the sandwichmethod, it is not always necessary that the antibody used for thelabeled antibody and for the solid phase should be one type or onespecies but a mixture of two or more antibodies may also be used for thepurpose of improving the assay sensitivity, etc.

[0681] In the method of assaying the peptide of the present invention bythe sandwich method according to the present invention, preferredmonoclonal antibodies of the present invention used for the primary andthe secondary reactions are antibodies, which binding sites to thepeptide of the present invention are different from each other. Thus,the antibodies used in the primary and secondary reactions are thosewherein, when the antibody used in the secondary reaction recognizes theC-terminal region of the peptide of the present invention, the antibodyrecognizing the site other than the C-terminal regions, e.g.,recognizing the N-terminal region, is preferably used in the primaryreaction.

[0682] The monoclonal antibody of the present invention may be used inan assay system other than the sandwich method, such as the competitivemethod, the immunometric method or the nephrometry.

[0683] In the competitive method, an antigen in a sample fluid and alabeled antigen are competitively reacted with an antibody, then anunreacted labeled antigen (F) and a labeled antigen bound to theantibody (B) are separated (i.e., B/F separation) and the labeled amountof either B or F is measured to determine the amount of the antigen inthe sample fluid. In the reactions for such a method, there are a liquidphase method in which a soluble antibody is used as the antibody and theB/F separation is effected by polyethylene glycol, while a secondantibody to the antibody is used, and a solid phase method in which animmobilized antibody is used as the first antibody or a soluble antibodyis used as the first antibody, while an immobilized antibody is used asthe second antibody.

[0684] In the immunometric method, an antigen in a sample fluid and animmobilized antigen are competitively reacted with a given amount of alabeled antibody followed by separating the solid phase from the liquidphase; or an antigen in a sample fluid and an excess amount of labeledantibody are reacted, then an immobilized antigen is added to bind anunreacted labeled antibody to the solid phase and the solid phase isseparated from the liquid phase. Thereafter, the labeled amount of anyof the phases is measured to determine the antigen amount in the samplefluid.

[0685] In the nephrometry, the amount of insoluble sediment, which isproduced as a result of the antigen-antibody reaction in a gel or in asolution, is measured. Even when the amount of an antigen in a samplefluid is small and only a small amount of the sediment is obtained, alaser nephrometry utilizing laser scattering can be suitably used.

[0686] In applying each of those immunoassays to the assay method of thepresent invention, any special conditions, operations, etc. are notrequired. The assay system for the peptide of the present invention maybe constructed in addition to conditions or operations conventionallyused for each of the methods, taking technical consideration by oneskilled in the art into account. For the details of such conventionaltechnical means, a variety of reviews, reference books, etc. may bereferred to:

[0687] for example, Hiroshi Irie (ed.): “Radioimmunoassay” (published byKodansha, 1974); Hiroshi Irie (ed.): “Radioimmunoassay; Second Series”(published by Kodansha, 1979); Eiji Ishikawa, et al. (ed.): “EnzymeImmunoassay” (published by Igaku Shoin, 1978); Eiji Ishikawa, et al.(ed.): “Enzyme Immunoassay” (Second Edition) (published by Igaku Shoin,1982); Eiji Ishikawa, et al. (ed.): “Enzyme Immunoassay” (Third Edition)(published by Igaku Shoin, 1987); “Methods in Enzymology” Vol. 70(Immunochemical Techniques (Part A)); ibid., Vol. 73 (ImmunochemicalTechniques (Part B)); ibid., Vol. 74 (Immunochemical Techniques (PartC)); ibid., Vol. 84 (Immunochemical Techniques (Part D: SelectedImmunoassays)); ibid., Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies)) (all published by Academic Press); etc.

[0688] As described above, the peptide of the present invention can bequantified with high sensitivity, using the antibody of the presentinvention.

[0689] Furthermore, when a reduced level of the peptide of the presentinvention is detected by quantifying a level of the peptide of thepresent invention using the antibody of the present invention, it can bediagnosed that one suffers from, e.g., anorexia, hypertension,autoimmune disease, heart failure, cataract, glaucoma, acute bacterialmeningitis, acute myocardial infarction, acute pancreatitis, acute viralencephalitis, adult respiratory distress syndrome, alcoholic hepatitis,Alzheimer's disease, asthma, arteriosclerosis, atopic dermatitis,bacterial pneumonia, bladder cancer, fracture, breast cancer, bulimia,polyphagia, burn healing, uterine cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic pancreatitis, livercirrhosis, cancer of the colon and rectum (colon cancer/rectal cancer),Crohn's disease, dementia, diabetic complications, diabetic nephropathy,diabetic neuropathy, diabetic retinopathy, gastritis, Helicobacterpylori bacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially, anorexia or the like) etc.; or itis highly likely for one to suffer from these disease in the future.

[0690] When an increased level of the peptide of the present inventionis detected, it can be diagnosed that one suffers from, e.g., obesity(e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma, Sheehan's syndrome,spermatogenesis disorder (especially, obesity, or the like), etc.; or itis highly likely for one to suffer from these disease in the future.

[0691] The antibody of the present invention may also be employed todetect the peptide of the present invention present in a sample fluidsuch as body fluids, tissues, etc. The antibody may further be used forthe preparation of an antibody column to purify the peptide of thepresent invention, detect the peptide of the present invention in eachfraction upon purification, analysis of the behavior of the peptide ofthe present invention in the cells under investigation.

[0692] (4) Gene Diagnostic Agent

[0693] By using the DNA of the present invention, e.g., as a probe,abnormality (gene abnormality) of the DNA or mRNA encoding the peptideof the present invention in human or other warm-blooded animal (e.g.,rat, mouse, guinea pig, rabbit, chicken, sheep, swine, bovine, horse,cat, dog, monkey, etc.), can be detected. Thus, the DNA of the presentinvention is useful as a gene diagnostic agent for the damage to the DNAor mRNA, mutation, a decreased expression or an increased expression, oroverexpression of the DNA or mRNA.

[0694] The gene diagnosis described above using the DNA of the presentinvention can be performed by, for example, the publicly known northernhybridization assay or the PCR-SSCP assay (Genomics, 5, 874-879 (1989);Proceedings of the National Academy of Sciences of the United States ofAmerica, 86, 2766-2770 (1989)).

[0695] When a decreased expression of mRNA is detected, e.g., bynorthern hybridization, it can be diagnosed that one is likely to sufferfrom, for example, anorexia, hypertension, autoimmune disease, heartfailure, cataract, glaucoma, acute bacterial meningitis, acutemyocardial infarction, acute pancreatitis, acute viral encephalitis,adult respiratory distress syndrome, alcoholic hepatitis, Alzheimer'sdisease, asthma, arteriosclerosis, atopic dermatitis, bacterialpneumonia, bladder cancer, fracture, breast cancer, bulimia, polyphagia,burn healing, uterine cervical cancer, chronic lymphocytic leukemia,chronic myelogenous leukemia, chronic pancreatitis, liver cirrhosis,cancer of the colon and rectum (colon cancer/rectal cancer), Crohn'sdisease, dementia, diabetic complications, diabetic nephropathy,diabetic neuropathy, diabetic retinopathy, gastritis, Helicobacterpylori bacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially anorexia or the like) etc.; or itis highly likely for one to suffer from diseases in the future.

[0696] When overexpression of mRNA is detected by northernhybridization, it can be diagnosed that one is likely to suffer from,for example, obesity (e.g., malignant mastocytosis, exogenous obesity,hyperinsulinar obesity, hyperplasmic obesity, hypophyseal adiposity,hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity,symptomatic obesity, infantile obesity, upper body obesity, alimentaryobesity, hypogonadal obesity, systemic mastocytosis, simple obesity,central obesity, etc.), hyperphagia, pituitary tumor, diencephalontumor, menstrual disorder, autoimmune disease, prolactinoma, sterility,impotence, amenorrhea, lactorrhea, acromegaly, Chiari-Frommel syndrome,Argonz-del Castillo syndrome, Forbes-Albright syndrome, lymphoma orSheehan's syndrome, spermatogenesis disorder, etc. (especially, obesityor the like); or it is highly likely for one to suffer from diseases inthe future.

[0697] (5) Pharmaceutical Composition Comprising Antisense DNA

[0698] The antisense DNA that binds complementarily to the DNA of thepresent invention to inhibit expression of the DNA can be used aspreventive/therapeutic agents for diseases, for example, obesity (e.g.,malignant mastocytosis, exogenous obesity, hyperinsulinar obesity,hyperplasmic obesity, hypophyseal adiposity, hypoplasmic obesity,hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc. (especially, obesity or the like), etc.

[0699] For example, when the antisense DNA is used, the antisense DNAmay be administered directly, or the DNA is inserted into an appropriatevector such as retrovirus vector, adenovirus vector,adenovirus-associated virus vector, etc. and then administered in aconventional manner. The antisense DNA may also be administered as anintact DNA, or prepared into pharmaceutical preparations together with aphysiologically acceptable carrier such as an adjuvant to assist itsuptake and administered by gene gun or through a catheter such as acatheter with a hydrogel.

[0700] In addition, the antisense DNA may also be employed as anoligonucleotide probe for diagnosis to examine the presence of the DNAof the present invention in tissues or cells and states of itsexpression.

[0701] (6) Pharmaceutical Composition Comprising the Antibody of thePresent Invention

[0702] The antibody of the present invention having the effect toneutralize the activity of the peptide of the present invention can beused as drugs for the prevention/treatment of diseases, for example,obesity (e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc. (especially, obesity, or the like), etc.

[0703] The therapeutic/preventive agents for diseases described abovecomprising the antibody of the present invention can be administered tohuman or mammals (e.g., rats, rabbits, sheep, swine, bovine, cats, dogs,monkeys, etc.) orally or parenterally directly as a liquid preparation,or as a pharmaceutical composition in an appropriate preparation form.The dose varies depending on subject to be administered, target disease,conditions, route for administration, etc.; when it is used for thetreatment/prevention of the adult patient with, e.g., obesity, the agentis advantageously administered to the patient through intravenousinjection, normally in a single dose of approximately 0.01 to 20 mg/kgbody weight, preferably about 0.1 to about 10 mg/kg body weight, andmore preferably about 0.1 to about 5 mg/kg body weight, approximately 1to 5 times, preferably approximately 1 to 3 times, per day. For otherparenteral administration and oral administration, the correspondingdose may be administered. When the conditions are extremely serious, thedose may be increased depending on the conditions.

[0704] The antibody of the present invention may be administereddirectly as it is or as an appropriate pharmaceutical composition. Thepharmaceutical composition used for the administration described abovecontains a pharmacologically acceptable carrier with the aforesaidcompounds or salts thereof, a diluent or excipient. Such a compositionis provided in the preparation suitable for oral or parenteraladministration.

[0705] That is, examples of the composition for oral administrationinclude solid or liquid preparations, specifically, tablets (includingdragees and film-coated tablets), pills, granules, powdery preparations,capsules (including soft capsules), syrup, emulsions, suspensions, etc.Such a composition is manufactured by publicly known methods andcontains a vehicle, a diluent or excipient conventionally used in thefield of pharmaceutical preparations. Examples of the vehicle orexcipient for tablets are lactose, starch, sucrose, magnesium stearate,etc.

[0706] Examples of the composition for parenteral administration thatcan be used are injections, suppositories, etc. and the injectionsinclude the form of intravenous, subcutaneous, transcutaneous,intramuscular and drip injections, etc. Such injections are prepared bypublicly known methods, e.g., by dissolving, suspending or emulsifyingthe aforesaid antibody or its salts in a sterile aqueous or oily liquidmedium. For the aqueous medium for injection, for example, physiologicalsaline and isotonic solutions containing glucose and other adjuvant,etc. are used. Appropriate dissolution aids, for example, alcohol (e.g.,ethanol), polyalcohol (e.g., propylene glycol or polyethylene glycol),nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50mol) adduct of hydrogenated castor oil)] may be used in combination. Forthe oily solution, for example, sesame oil, soybean oil and the like areused, and dissolution aids such as benzyl benzoate, benzyl alcohol, etc.may be used in combination. The thus-prepared liquid for injection isnormally filled in an appropriate ampoule. The suppository used forrectal administration is prepared by mixing the aforesaid antibody orits salts with conventional suppository base.

[0707] The oral or parenteral pharmaceutical composition described aboveis advantageously prepared in a unit dosage form suitable for the doseof the active ingredient. Examples of such unit dosage form includetablets, pills, capsules, injections (ampoules), suppositories, etc. Itis preferred that the antibody described above is contained generally ina dose of 5 to 500 mg per unit dosage form, 5 to 100 mg especially forinjections and 10 to 250 mg for other preparations.

[0708] Each composition described above may further contain other activecomponents unless formulation with the antibody causes any adverseinteraction.

[0709] (7) DNA Transgenic Animal

[0710] The present invention provides a non-human mammal bearing anexogenous DNA encoding the peptide of the present invention (hereinaftermerely referred to as the exogenous DNA of the present invention) or itsvariant DNA (sometimes simply referred to as the exogenous variant DNAof the present invention).

[0711] Thus, the present invention provides:

[0712] (i) a non-human mammal bearing the exogenous DNA or its variantDNA;

[0713] (ii) the mammal according to (i), wherein the non-human mammal isa rodent;

[0714] (iii) the mammal according to (ii), wherein the rodent is mouseor rat; and,

[0715] (iv) a recombinant vector bearing the exogenous DNA of thepresent invention or its variant DNA and capable of expressing in amammal.

[0716] The non-human mammal bearing the exogenous DNA of the presentinvention or its variant DNA (hereinafter simply referred to as the DNAtransgenic animal of the present invention) can be prepared bytransfecting a desired DNA into an unfertilized egg, a fertilized egg, aspermatozoon, a germinal cell containing a primordial germinal cellthereof, or the like, preferably in the embryogenic stage in thedevelopment of a non-human mammal (more preferably in the single cell orfertilized cell stage and generally before the 8-cell phase), bystandard means, such as the calcium phosphate method, the electric pulsemethod, the lipofection method, the agglutination method, themicroinjection method, the particle gun method, the DEAE-dextran methodetc. Also, it is possible to transfect the exogenous DNA of the presentinvention into a somatic cell, a living organ, a tissue cell, or thelike by the DNA transfection methods, and utilize the transformant forcell culture, tissue culture, etc. In addition, these cells may be fusedwith the above-described germinal cell by a publicly known cell fusionmethod to create the transgenic animal of the present invention.

[0717] Examples of the non-human mammal that can be used include bovine,swine, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice,rats, and the like. Above all, preferred are rodents, especially mice(e.g., C57BL/6 strain, DBA2 strain, etc. for a pure line and for a crossline, B6C3F₁ strain, BDF₁ strain B6D2F₁ strain, BALB/c strain, ICRstrain, etc.) or rats (Wistar, SD, etc.), since they are relativelyshort in ontogeny and life cycle from a standpoint of creating modelanimals for human disease.

[0718] “Mammals” in a recombinant vector that can be expressed in themammals include the aforesaid non-human mammals and human.

[0719] The exogenous DNA of the present invention refers to the DNA ofthe present invention that is once isolated/extracted from mammals, notthe DNA of the present invention inherently possessed by the non-humanmammals.

[0720] The variant DNA of the present invention includes variantsresulting from variation (e.g., mutation, etc.) in the base sequence ofthe original DNA of the present invention, specifically DNAs resultingfrom base addition, deletion, substitution with other bases, etc. andfurther including abnormal DNA.

[0721] The abnormal DNA is intended to mean such a DNA that expressesthe abnormal peptide of the present invention and exemplified by the DNAthat expresses a peptide to suppress the functions of the normal peptideof the present invention, or the like.

[0722] The exogenous DNA of the present invention may be any one ofthose derived from a mammal of the same species as, or a differentspecies from, the mammal as the target animal. In transfecting the DNAof the present invention, it is generally advantageous to use the DNA asa DNA construct in which the DNA is ligated downstream a promotercapable of expressing the DNA in the target animal. For example, in thecase of transfecting the human DNA of the present invention, a DNAtransgenic mammal that expresses the DNA of the present invention to ahigh level, can be prepared by microinjecting a DNA construct (e.g.,vector, etc.) ligated with the human DNA of the present invention into afertilized egg of the target non-human mammal downstream variouspromoters, which are capable of expressing the DNA derived from variousmammals (e.g., rabbits, dogs, cats, guinea pigs, hamsters, rats, mice,etc.) bearing the DNA of the present invention highly homologous to thehuman DNA.

[0723] As expression vectors for the peptide of the present invention,there are Escherichia coli-derived plasmids, Bacillus subtilis-derivedplasmids, yeast-derived plasmids, bacteriophages such as A phage,retroviruses such as Moloney leukemia virus, etc., and animal virusessuch as vaccinia virus, baculovirus, etc. Of these vectors, Escherichiacoli-derived plasmids, Bacillus subtilis-derived plasmids, oryeast-derived plasmids, etc. are preferably used.

[0724] Examples of these promoters for regulating the DNA expressiondescribed above include (1) promoters for the DNA derived from viruses(e.g., simian virus, cytomegalovirus, Moloney leukemia virus, JC virus,breast cancer virus, poliovirus, etc.), and (2) promoters derived fromvarious mammals (human, rabbits, dogs, cats, guinea pigs, hamsters,rats, mice, etc.), for example, promoters of albumin, insulin II,uroplakin II, elastase, erythropoietin, endothelin, muscular creatinekinase, glial fibrillary acidic protein, glutathione S-transferase,platelet-derived growth factor, keratins K1, K10 and K14, collagen typesI and II, cyclic AMP-dependent protein kinase βI subunit, dystrophin,tartarate-resistant alkaline phosphatase, atrial natriuretic factor,endothelial receptor tyrosine kinase (generally abbreviated as Tie2),sodium-potassium adenosine triphosphorylase (Na,K-ATPase), neurofilamentlight chain, metallothioneins I and IIA, metalloproteinase I tissueinhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamineβ-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor1α (EF-1α), β actin, α and β myosin heavy chains, myosin light chains 1and 2, myelin base protein, thyroglobulins, Thy-1, immunoglobulins,H-chain variable region (VNP), serum amyloid component P, myoglobin,troponin C, smooth muscle a actin, preproencephalin A, vasopressin, etc.Among others them, cytomegalovirus promoters, human peptide elongationfactor 1α (EF-1α) promoters, human and chicken β actin promoters etc.,which protein can highly express in the whole body are preferred.

[0725] It is preferred that the vectors described above have a sequencefor terminating the transcription of the desired messenger RNA in theDNA transgenic animal (generally called a terminator); for example, asequence of each DNA derived from viruses and various mammals. SV40terminator of simian virus, etc. are preferably used.

[0726] In addition, for the purpose of increasing the expression of thedesired exogenous DNA to a higher level, the splicing signal andenhancer region of each DNA, a portion of the intron of an eukaryoticDNA may also be ligated at the 5′ upstream of the promoter region, orbetween the promoter region and the translational region, or at the 3′downstream of the translational region, depending upon purpose.

[0727] The normal translational region for the peptide of the presentinvention can be obtained using as a starting material the entiregenomic DNA or its portion of liver, kidney, thyroid cell or fibroblastorigin from human or various mammals (e.g., rabbits, dogs, cats, guineapigs, hamsters, rats, mice, etc.) or of various commercially availablegenomic DNA libraries, or using complementary DNA prepared by a publiclyknown method from RNA of liver, kidney, thyroid cell or fibroblastorigin as a starting material. Also, an exogenous abnormal DNA canproduce a translational region, which is obtained by point mutagenesisvariation of the normal translational region for a peptide obtained fromthe cells or tissues described above.

[0728] The said translational region can be prepared by a conventionalDNA engineering technique, in which the DNA is ligated downstream theaforesaid promoter and if desired, upstream the translation terminationsite, as a DNA construct capable of being expressed in the transgenicanimal.

[0729] The exogenous DNA of the present invention is transfected at thefertilized egg cell stage in a manner such that the DNA is certainlypresent in all the germinal cells and somatic cells of the targetmammal. The fact that the exogenous DNA of the present invention ispresent in the germinal cells of the animal prepared by DNA transfectionmeans that all offspring of the prepared animal will maintain theexogenous DNA of the present invention in all of the germinal cells andsomatic cells thereof. The offspring of the animal that inherits theexogenous DNA of the present invention also have the exogenous DNA inall of the germinal cells and somatic cells thereof.

[0730] The non-human mammal, in which the normal exogenous DNA of thepresent invention has been transfected, can be passaged as theDNA-bearing animal under ordinary rearing environment, by confirmingthat the exogenous DNA is stably retained by mating.

[0731] By the transfection of the exogenous DNA of the present inventionat the fertilized egg cell stage, the DNA is retained to be excess inall of the germinal and somatic cells. The fact that the exogenous DNAof the present invention is excessively present in the germinal cells ofthe prepared animal after transfection means that the exogenous DNA ofthe present invention is excessively present in all of the germinalcells and somatic cells thereof. The offspring of the animal thatinherits the exogenous DNA of the present invention have excessively theexogenous DNA of the present invention in all of the germinal cells andsomatic cells thereof.

[0732] By obtaining a homozygotic animal having the transfected DNA inboth of homologous chromosomes and mating a male and female of theanimal, all offspring can be passaged to retain the DNA.

[0733] In a non-human mammal bearing the normal DNA of the presentinvention, the normal DNA of the present invention has expressed to ahigh level, and may eventually develop the hyperfunction of the peptideof the present invention by promoting the function of endogenous normalDNA. Therefore, the animal can be utilized as a pathologic model animalfor such a disease. Specifically, using the normal DNA transgenic animalof the present invention, it is possible to elucidate the mechanism ofthe hyperfunction of the peptide of the present invention and thepathological mechanism of the disease associated with the peptide of thepresent invention and to determine how to treat the disease.

[0734] Furthermore, since a mammal wherein the exogenous normal DNA ofthe present invention is transfected exhibits an increasing symptom ofthe peptide of the present invention librated, the animal is usable forscreening therapeutic agents for the disease associated with the peptideof the present invention.

[0735] On the other hand, a non-human mammal having the exogenousabnormal DNA of the present invention can be passaged under normalbreeding conditions as the DNA-bearing animal by confirming the stableretaining of the exogenous DNA via crossing. Further, the exogenous DNAto be subjected can be utilized as a starting material by inserting theDNA into the plasmid described above. The DNA construct with promotercan be prepared by conventional DNA engineering techniques. Thetransfection of the abnormal DNA of the present invention at thefertilized egg cell stage is preserved to be present in all of thegerminal and somatic cells of the mammals to be subjected. The fact thatthe abnormal DNA of the present invention is present in the germinalcells of the animal after DNA transfection means that all of theoffspring of the prepared animal have the abnormal DNA of the presentinvention in all of the germinal and somatic cells. Such an offspringpassaged the exogenous DNA of the present invention contains theabnormal DNA of the present invention in all of the germinal and somaticcells. A homozygous animal having the introduced DNA on both ofhomologous chromosomes can be acquired and then by mating these male andfemale animals, all the offspring can be bled to have the DNA.

[0736] Since a non-human mammal having the abnormal DNA of the presentinvention may express the abnormal DNA of the present invention at ahigh level, the animal may be the function inactivation typeinadaptability of the peptide of the present invention by inhibiting thefunction of the endogenous normal DNA and can be utilized as its diseasemodel animal. For example, using the abnormal DNA-transgenic animal ofthe present invention, it is possible to elucidate the mechanism ofinadaptability of the peptide of the present invention and to perform tostudy a method for treatment of this disease.

[0737] More specifically, the transgenic animal of the present inventionexpressing the abnormal DNA of the present invention to a high level isalso expected to serve as an experimental model for the elucidation ofthe mechanism of the functional inhibition (dominant negative effect) ofnormal peptide by the abnormal peptide of the present invention in thefunction inactive type inadaptability of the peptide of the presentinvention.

[0738] A mammal bearing the abnormal exogenous DNA of the presentinvention is also expected to serve for screening a candidate drug forthe treatment of the function inactive type inadaptability of thepeptide of the present invention, since the peptide of the presentinvention is increased in such an animal in its free form.

[0739] Other potential applications of two kinds of the transgenicanimals described above include:

[0740] (1) use as a cell source for tissue culture;

[0741] (2) elucidation of the relation to a peptide that is specificallyexpressed or activated by the peptide of the present invention, bydirect analysis of DNA or RNA in tissue of the DNA transgenic animal ofthe present invention or by analysis of the peptide tissue expressed bythe DNA;

[0742] (3) research in the function of cells derived from tissues thatare cultured usually only with difficulty, using cells of tissue bearingthe DNA cultured by a standard tissue culture technique;

[0743] (4) screening of a drug that enhances the functions of cellsusing the cells described in (3) above; and,

[0744] (5) isolation and purification of the variant peptide of thepresent invention and preparation of an antibody thereto.

[0745] Furthermore, clinical conditions of a disease associated wit thepeptide of the present invention, including the function inactive typeinadaptability of the peptide of the present invention can be determinedusing the DNA transgenic animal of the present invention. Also,pathological findings on each organ in a disease model associated withthe peptide of the present invention can be obtained in more detail,leading to the development of a new method for treatment as well as theresearch and therapy of any secondary diseases associated with thedisease.

[0746] It is also possible to obtain a free DNA-transfected cell bywithdrawing each organ from the DNA transgenic animal of the presentinvention, mincing the organ and degrading with a proteinase such astrypsin, etc., followed by establishing the line of culturing orcultured cells. Furthermore, the DNA transgenic animal of the presentinvention can serve as identification of cells capable of producing thepeptide of the present invention, and as studies on association withapoptosis, differentiation or propagation or on the mechanism of signaltransduction in these properties to inspect any abnormality therein.Thus, the DNA transgenic animal of the present invention can provide aneffective research material for the peptide of the present invention andfor elucidating the function and effect thereof.

[0747] To develop a therapeutic drug for the treatment of diseasesassociated with the peptide of the present invention, including thefunction inactive type inadaptability of the peptide of the presentinvention, using the DNA transgenic animal of the present invention, aneffective and rapid method for screening can be provided by using themethod for inspection and the method for quantification, etc. describedabove. It is also possible to investigate and develop a method for DNAtherapy for the treatment of diseases associated with the peptide of thepresent invention, using the DNA transgenic animal of the presentinvention or a vector capable of expressing the exogenous DNA of thepresent invention.

[0748] (8) Knockout Animal

[0749] The present invention provides a non-human mammal embryonic stemcell bearing the DNA of the present invention inactivated and anon-human mammal deficient in expressing the DNA of the presentinvention.

[0750] Thus, the present invention provides:

[0751] (i) a non-human embryonic stem cell in which the DNA of thepresent invention is inactivated;

[0752] (ii) an embryonic stem cell according to (i), wherein the DNA isinactivated by introducing a reporter gene (e.g., β-galactosidase genederived from Escherichia coli);

[0753] (iii) an embryonic stem cell according to (i), which is resistantto neomycin;

[0754] (iv) an embryonic stem cell according to (i), wherein thenon-human mammal is a rodent;

[0755] (v) an embryonic stem cell according to (iv), wherein the rodentis mouse;

[0756] (vi) a non-human mammal deficient in expressing the DNA of thepresent invention, wherein the DNA of the present invention isinactivated;

[0757] (vii) a non-human mammal according to (vi), wherein the DNA isinactivated by inserting a reporter gene (e.g., β-galactosidase derivedfrom Escherichia coli) therein and the reporter gene is capable of beingexpressed under control of a promoter for the DNA of the presentinvention;

[0758] (viii) a non-human mammal according to (vi), which is a rodent;

[0759] (ix) a non-human mammal according to (viii), wherein the rodentis mouse; and,

[0760] (x) a method of screening a compound or its salt that promotes orinhibits the promoter activity for the DNA of the present invention,which comprises administering a test compound to the mammal of (vii) anddetecting expression of the reporter gene.

[0761] The non-human mammal embryonic stem cell in which the DNA of thepresent invention is inactivated refers to a non-human mammal embryonicstem cell that suppresses the ability of the non-human mammal to expressthe DNA by artificially mutating the DNA of the present invention, orthe DNA has no substantial ability to express the peptide of the presentinvention (hereinafter sometimes referred to as the knockout DNA of thepresent invention) by substantially inactivating the activity of thepeptide of the present invention encoded by the DNA (hereinafter merelyreferred to as ES cell).

[0762] As the non-human mammal, the same examples as described aboveapply.

[0763] Techniques for artificially mutating the DNA of the presentinvention include deletion of a part or all of the DNA sequence andinsertion of or substitution with other DNA, by genetic engineering. Bythese variations, the knockout DNA of the present invention may beprepared, for example, by shifting the reading frame of a codon or bydisrupting the function of a promoter or exon.

[0764] Specifically, the non-human mammal embryonic stem cell in whichthe DNA of the present invention is inactivated (hereinafter merelyreferred to as the ES cell with the DNA of the present inventioninactivated or the knockout ES cell of the present invention) can beobtained by, for example, isolating the DNA of the present inventionthat the desired non-human mammal possesses, inserting a DNA fragmenthaving a DNA sequence constructed by inserting a drug resistant genesuch as a neomycin resistant gene or a hygromycin resistant gene, or areporter gene such as lacZ (β-galactosidase gene) or cat(chloramphenicol acetyltransferase gene), etc. into its exon sitethereby to disable the functions of exon, or integrating to a chromosomeof the subject animal by, e.g., homologous recombination, a DNA sequencewhich terminates gene transcription (e.g., polyA additional signal,etc.) in the intron between exons, thus inhibiting the synthesis ofcomplete messenger RNA to eventually destroy the gene (hereinaftersimply referred to as targeting vector). The thus obtained ES cells aresubjected to Southern hybridization analysis using a DNA sequence on ornear the DNA of the present invention as a probe, or to PCR analysisusing a DNA sequence on the targeting vector and another DNA sequencenear the DNA of the present invention, which is not included in thetargeting vector as primers, thereby to select the knockout ES cell ofthe present invention.

[0765] The parent ES cells to inactivate the DNA of the presentinvention by homologous recombination, etc. may be of a strain alreadyestablished as described above, or may be originally established inaccordance with a modification of the known method by Evans and Kaufmansupra. For example, in the case of mouse ES cells, currently it iscommon practice to use ES cells of the 129 strain. However, since theirimmunological background is obscure, the C57BL/6 mouse or the BDF₁ mouse(F₁ hybrid between C57BL/16 and DBA/2), wherein the low ovumavailability per C57BL/6 in the C57B116 mouse has been improved bycrossing with DBA/2, may be preferably used, instead of obtaining a pureline of ES cells with the clear immunological genetic background and forother purposes. The BDF₁ mouse is advantageous in that, when apathologic model mouse is generated using ES cells obtained therefrom,the genetic background can be changed to that of the C57BL/6 mouse byback-crossing with the C57BL/6 mouse, since its background is of theC57BL/6 mouse, as well as being advantageous in that ovum availabilityper animal is high and ova are robust.

[0766] In establishing ES cells, blastocytes at 3.5 days afterfertilization are commonly used. In the present invention, embryos arepreferably collected at the 8-cell stage, after culturing until theblastocyte stage, the embryos are used to efficiently obtain a largenumber of early stage embryos.

[0767] Although the ES cells used may be of either sex, male ES cellsare generally more convenient for generation of a germ cell line chimeraand are therefore preferred. It is also desirable that sexes areidentified as soon as possible to save painstaking culture time.

[0768] Methods for sex identification of the ES cell include the methodin which a gene in the sex-determining region on the Y-chromosome isamplified by the PCR process and detected. When this method is used, onecolony of ES cells (about 50 cells) is sufficient for sex-determinationanalysis, which karyotype analysis, for example G-banding method,requires about 10⁶ cells; therefore, the first selection of ES cells atthe early stage of culture can be based on sex identification, and malecells can be selected early, which saves a significant amount of time atthe early stage of culture.

[0769] Second selection can be achieved by, for example, number ofchromosome confirmation by the G-banding method. It is usually desirablethat the chromosome number of the obtained ES cells be 100% of thenormal number. However, when it is difficult to obtain the cells havingthe normal number of chromosomes due to physical operation etc. in cellestablishment, it is desirable that the ES cell be again cloned to anormal cell (e.g., in mouse cells having the number of chromosomes being2n=40) after the gene of the ES cells is rendered knockout.

[0770] Although the embryonic stem cell line thus obtained shows a veryhigh growth potential, it must be subcultured with great care, since ittends to lose its ontogenic capability. For example, the embryonic stemcell line is cultured at about 37° C. in a carbon dioxide incubator(preferably about 5% carbon dioxide and about 95% air, or about 5%oxygen, about 5% carbon dioxide and 90% air) in the presence of LIF(1-10000 U/ml) on appropriate feeder cells such as STO fibroblasts,treated with a trypsin/EDTA solution (normally about 0.001 to about 0.5%trypsin/about 0.1 to about 5 mM EDTA, preferably about 0.1% trypsin/1 mMEDTA) at the time of passage to obtain separate single cells, which arethen seeded on freshly prepared feeder cells. This passage is normallyconducted every 1 to 3 days; it is desirable that cells are observed atpassage and cells found to be morphologically abnormal in culture, ifany, should be abandoned.

[0771] Where ES cells are allowed to reach a high density in mono-layersor to form cell aggregates in suspension under appropriate conditions,they will spontaneously differentiate to various cell types, forexample, pariental and visceral muscles, cardiac muscle or the like (M.J. Evans and M. H. Kaufman, Nature, 292, 154, 1981; G. R. Martin, Proc.Natl. Acad. Sci. U.S.A., 78, 7634, 1981; T. C. Doetschman et al.,Journal of Embryology Experimental Morphology, 87, 27, 1985). The cellsdeficient in expressing the DNA of the present invention, which areobtainable from the differentiated ES cells of the present invention,are useful for studying the peptide of the present invention or thereceptor protein of the present invention from an aspect of cellbiology.

[0772] The non-human mammal deficient in expressing the DNA of thepresent invention can be identified from a normal animal by measuringthe mRNA amount in the subject animal by a publicly known method, andindirectly comparing the levels of expression.

[0773] As the non-human mammal, the same examples as described aboveapply.

[0774] With respect to the non-human mammal deficient in expressing theDNA of the present invention, the DNA of the present invention can berendered knockout by transfecting a targeting vector, prepared asdescribed above, to non-human mammal embryonic stem cells or oocytesthereof, and conducting homologous recombination in which a targetingvector DNA sequence, wherein the DNA of the present invention isinactivated by the transfection, is replaced with the DNA of the presentinvention on a chromosome of a non-human mammal embryonic stem cell orembryo thereof.

[0775] The cells with the DNA of the present invention knockout can beidentified by Southern hybridization analysis using a DNA sequence on ornear the DNA of the present invention as a probe, or by PCR analysisusing as primers a DNA sequence on the targeting vector and another DNAsequence, which is not included in the targeting vector. When non-humanmammalian embryonic stem cells are used, a cell line wherein the DNA, ofthe present invention is inactivated by homologous recombination iscloned; the resulting cloned cell line is injected to, e.g., a non-humanmammalian embryo or blastocyte, at an appropriate stage such as the8-cell stage. The resulting chimeric embryos are transplanted to theuterus of the pseudopregnant non-human mammal. The resulting animal is achimeric animal composed of both cells having the normal locus of theDNA of the present invention and those having an artificially mutatedlocus of the DNA of the present invention.

[0776] When some germ cells of the chimeric animal have a mutated locusof the DNA of the present invention, an individual, which entire tissueis composed of cells having a mutated locus of the DNA of the presentinvention can be selected from a series of offspring obtained bycrossing between such a chimeric animal and a normal animal, e.g., bycoat color identification, etc. The individuals thus obtained arenormally deficient in heterozygous expression of the peptide of thepresent invention. The individuals deficient in homozygous expression ofthe peptide of the present invention or the receptor protein of thepresent invention can be obtained from offspring of the intercrossbetween the heterozygotes of the peptide of the present invention or thereceptor protein of the present invention.

[0777] When an oocyte or egg cell is used, a DNA solution may beinjected, e.g., to the prenucleus by microinjection thereby to obtain atransgenic non-human mammal having a targeting vector introduced in achromosome thereof. From such transgenic non-human mammals, those havinga mutation at the locus of the DNA of the present invention can beobtained by selection based on homologous recombination.

[0778] As described above, individuals in which the DNA of the presentinvention is rendered knockout permit passage rearing under ordinaryrearing conditions, after the individuals obtained by their crossinghave proven to have been knockout.

[0779] Furthermore, the genital system may be obtained and maintained byconventional methods. That is, by crossing male and female animals eachhaving the inactivated DNA, homozygote animals having the inactivatedDNA in both loci can be obtained. The homozygotes thus obtained may bereared so that one normal animal and two or more homozygotes areproduced from a mother animal to efficiently obtain such homozygotes. Bycrossing male and female heterozygotes, homozygotes and heterozygoteshaving the inactivated DNA are proliferated and passaged.

[0780] The non-human mammal embryonic stem cell, in which the DNA of thepresent invention is inactivated, is very useful for preparing anon-human mammal deficient in expressing the DNA of the presentinvention.

[0781] Since the non-human mammal deficient in expressing the DNA of thepresent invention lacks various biological activities derived from thepeptide of the present invention, such an animal can be a disease modelsuspected of inactivated biological activities of the peptide of thepresent invention and thus, offers an effective study to investigatecauses for and therapy for these diseases.

[0782] (8a) Method of Screening Compounds having Therapeutic/PreventiveEffects on Diseases caused by Deficiency, Damages, etc. of the DNA ofthe Present Invention

[0783] The non-human mammal deficient in expressing the DNA of thepresent invention can be employed for screening of compounds havingtherapeutic/prophylactic effects on diseases caused by deficiency,damages, etc. of the DNA of the present invention.

[0784] That is, the present invention provides a method for screening ofa compound or its salt having therapeutic/preventive effects on diseasescaused by deficiency, damages, etc. of the DNA of the present invention,which comprises administering a test compound to the non-human mammaldeficient in expressing the DNA of the present invention andobserving/measuring a change occurred in the animal.

[0785] As the non-human mammal deficient in expressing the DNA of thepresent invention which can be employed for the screening method, thesame examples as given hereinabove apply.

[0786] Examples of the test compounds include peptides, proteins,non-peptide compounds, synthetic compounds, fermentation products, cellextracts, vegetable extracts, animal tissue extracts, blood plasma, etc.These compounds may be novel compounds or publicly known compounds.

[0787] Specifically, the non-human mammal deficient in expressing theDNA of the present invention is treated with a test compound, comparisonis made with an intact animal for control and a change in each organ,tissue, disease conditions, etc. of the animal is used as an indicatorto assess the therapeutic/prophylactic effects of the test compound.

[0788] For treating an animal to be test with a test compound, forexample, oral administration, intravenous injection, etc. are appliedand the treatment is appropriately selected depending upon conditions ofthe test animal, properties of the test compound, etc. Furthermore, adose of test compound to be administered can be appropriately chosendepending on method for administration, nature of the test compound,etc.

[0789] In screening compounds having the therapeutic/preventive effecton, e.g., anorexia, hypertension, autoimmune disease, heart failure,cataract, glaucoma, acute bacterial meningitis, acute myocardialinfarction, acute pancreatitis, acute viral encephalitis, adultrespiratory distress-syndrome, alcoholic hepatitis, Alzheimer's disease,asthma, arteriosclerosis, atopic dermatitis, bacterial pneumonia,bladder cancer, fracture, breast cancer, bulimia, polyphagia, burnhealing, uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease, etc. (especially, anorexia, or the like), thenon-human mammal deficient in expressing the DNA of the presentinvention is subjected to a sugar loading treatment, a test compound isadministered before or after the sugar loading treatment and, bloodsugar level, body weight change, etc. of the animal is measured withpassage of time.

[0790] In the screening method described above, when a test compound isadministered to a test animal and found to reduce the blood sugar levelof the animal to at least about 10%, preferably at least about 30% andmore preferably at least 25 about 50%, the test compound can be selectedto be a compound having a therapeutic/preventive effect on the diseasesabove.

[0791] The compound obtained using the screening method above is acompound selected from the test compounds described, above and exhibitsa therapeutic/preventive effect on the diseases caused by deficiencies,damages, etc. of the peptide of the present invention. Therefore, thecompound can be employed as a safe and low toxic drug for the treatmentand prevention of these diseases. Furthermore, compounds derived fromsuch a compound obtained by the screening described above can besimilarly employed.

[0792] The compound obtained by the screening method above may be in theform of salts. As such salts, there may be used salts withphysiologically acceptable acids (e.g., inorganic acids, organic acids,etc.) or bases (e.g., alkali metal salts, etc.), preferably in the formof physiologically acceptable acid addition salts. Examples of suchsalts are salts with inorganic acids (e.g., hydrochloric acid,phosphoric acid, hydrobromic acid, sulfuric acid, etc.), salts withorganic acids (e.g., acetic acid, formic acid, propionic acid, fumaricacid, maleic acid, succinic acid, tartaric acid, citric acid, malicacid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonicacid, etc.) and the like.

[0793] A pharmaceutical composition comprising the compound or its salt,obtained by the above screening method, may be manufactured in a mannersimilar to the method for preparing the pharmaceutical compositioncomprising the peptide of the present invention described hereinabove.

[0794] Since the pharmaceutical composition thus obtained is safe andlow toxic, it can be administered to human or mammals (e.g., rats, mice,guinea pigs, rabbits, sheep, swine, bovine, horses, cats, dogs, monkeys,etc.).

[0795] A dose of the compound or its salt to be administered variesdepending upon particular disease, subject to be administered, route ofadministration, etc., and in oral administration to an adult patientwith anorexia (as 60 kg body weight), the compound is administeredgenerally in a dose of approximately 0.1 to 100 mg, preferablyapproximately 1.0 to 50 mg, more preferably approximately 1.0 to 20 mgper day. For parenteral administration to an adult patient with anorexia(as 60 kg body weight), it is advantageous to administer the compoundintravenously in the form of an injectable preparation in a dose ofapproximately 0.01 to 30 mg, preferably approximately 0.1 to 20 mg, morepreferably approximately 0.1 to 10 mg per day, though the single dosagevaries depending upon particular subject, particular disease, etc. Forother animals, the compound can be administered in the correspondingdose with converting it into that for the 60 kg body weight.

[0796] (8b) Method of Screening a Compound that Promotes or Inhibits theActivity of a Promoter to the DNA of the Present Invention

[0797] The present invention provides a method of screening a compoundor its salt that promotes or inhibits the activity of a promoter to theDNA of the present invention, which comprises administering a testcompound to a non-human mammal deficient in expressing the DNA of thepresent invention and detecting expression of the reporter gene.

[0798] In the screening method described above, the non-human mammaldeficient in expressing the DNA of the present invention is selectedfrom the aforesaid non-human mammal deficient in expressing the DNA ofthe present invention, as an animal in which the DNA of the presentinvention is inactivated by introducing a reporter gene and the reportergene is expressed under control of a promoter to the DNA of the presentinvention.

[0799] The same examples of the test compound apply to those givenabove.

[0800] As the reporter gene, the same specific examples apply.Preferably employed are β-galactosidase (lacZ), soluble alkalinephosphatase gene, luciferase gene and the like.

[0801] Since the reporter gene is present under control of a promoter tothe DNA of the present invention in the non-human mammal deficient inexpressing the DNA of the present invention wherein the DNA of thepresent invention is substituted with the reporter gene, the activity ofthe promoter can be detected by tracing expression of a substanceencoded by the reporter gene.

[0802] For example, when a part of the DNA region encoding the peptideof the present invention is substituted with, e.g., β-galactosidase gene(lacZ) derived from Escherichia coli, β-galactosidase is expressed in atissue where the peptide of the present invention should originally beexpressed, instead of the peptide of the present invention. Thus, thestate of expression condition of the peptide of the present inventioncan be readily observed in vivo of an animal by staining with a reagent,e.g., 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal) which issubstrate for β-galactosidase. Specifically, a mouse deficient in thepeptide of the present invention, or its tissue slice section is fixedwith glutaraldehyde, etc. After washing with phosphate buffered saline(PBS), the system is reacted with a staining solution containing X-galat room temperature or about 37° C. for approximately 30 minutes to anhour. After the β-galactosidase reaction is terminated by washing thetissue preparation with 1 mM EDTA/PBS solution, the color formed isobserved. Alternatively, mRNA encoding lacZ may be detected in aconventional manner.

[0803] The compound or salts thereof obtained using the aforesaidscreening method are compounds that are selected from the test compoundsdescribed above and the compounds that promote or inhibit the activityof a promoter to the DNA of the present invention.

[0804] The compound obtained by the screening method above may formsalts. As salts of the compound, there may be used salts withphysiologically acceptable acids (e.g., inorganic acids, etc.) or bases(e.g., organic acids, etc.), and especially preferred arephysiologically acceptable acid addition salts. Examples of such saltsare salts with inorganic acids (e.g., hydrochloric acid, phosphoricacid, hydrobromic acid, sulfuric acid, etc.), salts with organic acids(e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleicacid, succinic acid, tartaric acid, citric acid, malic acid, oxalicacid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.)and the like.

[0805] The compound or its salt that promotes the promoter activity tothe DNA of the present invention can promote expression of the peptideof the present invention thereby to promote the function of the peptide.Thus, these compounds are useful as safe and low-toxic drugs for thetreatment/prevention of diseases, e.g., anorexia, hypertension,autoimmune disease, heart failure, cataract, glaucoma, acute bacterialmeningitis, acute myocardial infarction, acute pancreatitis, acute viralencephalitis, adult respiratory distress syndrome, alcoholic hepatitis,Alzheimer's disease, asthma, arteriosclerosis, atopic dermatitis,bacterial pneumonia, bladder cancer, fracture, breast cancer, bulimia,polyphagia, burn healing, uterine cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic pancreatitis, livercirrhosis, cancer of the colon and rectum (colon cancer/rectal cancer),Crohn's disease, dementia, diabetic complications, diabetic nephropathy,diabetic neuropathy, diabetic retinopathy, gastritis, Helicobacterpylori bacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease, etc. (especially, anorexia or the like) orthe like (especially, appetite (eating) stimulant).

[0806] The compound or its salt that inhibits the activity of a promoterto the DNA of the present invention can inhibit expression of thepeptide of the present invention thereby to inhibit the function of thepeptide. Thus, these compounds are useful as drugs, includingpreventive/therapeutic drugs (prolactin production inhibitors) fordiseases, for example, obesity (e.g., malignant mastocytosis, exogenousobesity, hyperinsulinar obesity, hyperplasmic obesity, hypophysealadiposity, hypoplasmic obesity, hypothyroid obesity, hypothalamicobesity, symptomatic obesity, infantile obesity, upper body obesity,alimentary obesity, hypogonadal obesity, systemic mastocytosis, simpleobesity, central obesity, etc.), hyperphagia, pituitary tumor,diencephalon tumor, menstrual disorder, autoimmune disease,prolactinoma, sterility, impotence, amenorrhea, lactorrhea, acromegaly,Chiari-Frommel syndrome, Argonz-del Castillo syndrome, Forbes-Albrightsyndrome, lymphoma or Sheehan's syndrome, spermatogenesis disorders,etc.; preferably as preventive/therapeutic agents for obesity,hyperphagia, etc.

[0807] Furthermore, compounds derived from the compounds obtained by thescreening described above may be likewise used.

[0808] The pharmaceuticals comprising the compound or its salt obtainedby the screening method may be manufactured as in the aforesaidpharmaceuticals comprising the peptide of the present invention or itssalt.

[0809] Since the pharmaceutical preparation thus obtained is safe andlow toxic, it can be administered to human or mammals (e.g., rats, mice,guinea pigs, rabbits, sheep, swine, bovine, horses, cats, dogs, monkeys,etc.).

[0810] A dose of the compound or its salt to be administered variesdepending upon target disease, subject to be administered, route ofadministration, etc.; when the compound that promotes the promoteractivity to the DNA of the present invention is orally administered toan adult patient with, e.g., anorexia (as 60 kg body weight), thecompound is administered generally in a dose of approximately 0.1 to 100mg, preferably approximately 1.0 to 50 mg, more preferably approximately1.0 to 20 mg per day. In parenteral administration, a single dose of thecompound varies depending upon subject to be administered, targetdisease, etc. When the compound that promotes the promoter activity tothe DNA of the present invention is administered to an adult patientwith, e.g., anorexia (as 60 kg body weight) in the form of an injectablepreparation, it is advantageous to administer the compound,intravenously in a dose of approximately 0.01 to 30 mg, preferablyapproximately 0.1 to 20 mg, more preferably approximately 0.1 to 10 mgper day. For other animals, the compound can be administered in thecorresponding dose with converting it into that for the 60 kg bodyweight.

[0811] On the other hand, when a compound that inhibits the promoteractivity to the DNA of the present invention is orally administered, thecompound is orally administered to an adult patient with, e.g., obesity(as 60 kg body weight) generally in a dose of approximately 0.1 to 100mg, preferably approximately 1.0 to 50 mg, more preferably approximately1.0 to 20 mg per day. In parenteral administration, a single dose of thecompound varies depending upon subject to be administered, targetdisease, etc. When the compound that inhibits the promoter activity tothe DNA of the present invention is administered to an adult patientwith, e.g., obesity (as 60 kg body weight) in the form of an injectablepreparation, it is advantageous to administer the compound intravenouslyin a dose of approximately 0.01 to 30 mg, preferably approximately 0.1to 20 mg, more preferably approximately 0.1 to 10 mg per day. For otheranimals, the compound can be administered in the corresponding dose withconverting it into that for the 60 kg body weight.

[0812] As described above, the non-human mammal deficient in expressingthe DNA of the present invention is extremely useful for screening acompound or its salt that promotes or inhibits the activity of apromoter to the DNA of the present invention, and can thus greatlycontribute to investigations of causes for various diseases caused byfailure to express the DNA of the present invention or to development ofpreventive/therapeutic agents for these diseases.

[0813] Moreover, when a so-called transgenic animal (gene-transfectedanimal) is prepared by using a DNA containing the promoter region of thepeptide of the present invention, ligating genes encoding variousproteins downstream the same and injecting the genes into animal oocyte,the peptide can be specifically synthesized by the animal so that itbecomes possible to investigate the activity in vivo. Furthermore, whenan appropriate reporter gene is ligated to the promoter region describedabove to establish a cell line so as to express the gene, such can beused as a survey system of low molecular weight compounds thatspecifically promotes or suppresses the ability of producing the peptideitself of the present invention in vivo.

[0814] The utilities of bovine GPR7 and bovine GPR8 (hereinafter merelyreferred to as the bovine GPR7/8) of the present invention are explainedbelow.

[0815] Antibodies to the bovine GPR7/8 of the present invention(hereinafter sometimes simply referred to as the bovine GPR7/8 antibody)may be any of polyclonal antibodies and monoclonal antibodies, as longas they are capable of recognizing antibodies to the bovine GPR7/8 ofthe present invention.

[0816] The antibody of the present invention may be manufactured bypublicly known methods for manufacturing antibodies or antisera, usingthe bovine GPR7/8 of the present invention as an antigen.

[0817] The antisense DNA (hereinafter sometimes merely referred to asthe bovine GPR7/8 antisense DNA) having a complementary or substantiallycomplementary base sequence to the DNA (hereinafter sometimes merelyreferred to as the bovine GPR7/8 DNA) encoding the bovine GPR7/8 of thepresent invention can be any antisense DNA, so long as it has a basesequence complementary or substantially complementary to that of thebovine GPR7/8 DNA of the present invention and capable of suppressingexpression of the DNA.

[0818] The base sequence substantially complementary to the bovineGPR7/8 DNA of the present invention may, for example, be a base sequencehaving at least about 70% homology, preferably at least about 80%homology, more preferably at least about 90% homology and mostpreferably at least about 95% homology, to the full-length base sequenceor partial base sequence of the base sequence complementary to thebovine GPR7/8 DNA of the present invention (i.e., complementary strandto the DNA of the present invention). In the entire base sequence of thecomplementary strand to the bovine GPR7/8 DNA of the present invention,an antisense DNA having at least about 70% homology, preferably at leastabout 80% homology, more preferably at least about 90% homology and mostpreferably at least about 95% homology, to the complementary strand ofthe base sequence which encodes the N-terminal region of the bovineGPR7/8 of the present invention (e.g., the base sequence around theinitiation codon) is particularly preferred. These antisense DNAs can besynthesized using a publicly known DNA synthesizer, etc.

[0819] The utilities of (1) the bovine GPR7/8 of the present invention,(2) the bovine GPR7/8 DNA of the present invention, (3) the bovineGPR7/8 antibody of the present invention, and (4) the bovine GPR7/8antisense DNA are explained below.

[0820] (1) Therapeutic/Preventive Agent for Diseases with which theBovine GPR7/8 of the Present Invention is Associated

[0821] As shown in EXAMPLE 25 later described, the bovine GPR7/8 of thepresent invention is a receptor to the peptide of the present invention.

[0822] Accordingly, when the bovine GPR7/8 of the present invention orthe bovine GPR7/8 DNA of the present invention involves any abnormalityor deficiency, it is highly likely to cause various diseases, includinganorexia, hypertension, autoimmune disease, heart failure, cataract,glaucoma, acute bacterial meningitis, acute myocardial infarction, acutepancreatitis, acute viral encephalitis, adult respiratory distresssyndrome, alcoholic hepatitis, Alzheimer's disease, asthma,arteriosclerosis, atopic dermatitis, bacterial pneumonia, bladdercancer, fracture, breast cancer, bulimia, polyphagia, burn healing,uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type I), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative diseases (especially anorexia, etc.), or the like.

[0823] Therefore, the bovine GPR7/8 of the present invention and thebovine GPR7/8 DNA of the present invention can be used aspharmaceuticals (in particular, appetite (eating) stimulants, etc.) forthe treatment/prevention of various diseases as described above(especially anorexia, etc.).

[0824] When a patient has a reduced level of, or deficient in the bovineGPR7/8 of the present invention in his or her body, the bovine GPR7/8 ofthe present invention and the bovine GPR7/8 DNA of the present inventioncan exhibit the role of the bovine GPR7/8 of the present inventionsufficiently or properly for the patient, (a) by administering thebovine GPR7/8 DNA of the present invention to the patient to express thebovine GPR7/8 of the present invention in the body, (b) by inserting thebovine GPR7/8 DNA of the present invention into a cell, expressing thebovine GPR7/8 of the present invention and then transplanting the cellto the patient, or (c) by administering the bovine GPR7/8 of the presentinvention to the patient, or the like.

[0825] Where the bovine GPR7/8 of the present invention or the bovineGPR7/8 DNA of the present invention is used as drugs for thetreatment/prevention described above, these drugs can be manufacturedand used similarly to the pharmaceuticals comprising the peptide of thepresent invention or the DNA of the present invention described above.

[0826] (2) Screening of Drug Candidate Compounds for Diseases

[0827] (2-1) Screening Method A

[0828] The method of screening a compound or its salt that alters thebinding of the bovine GPR7/8 of the present invention to the peptide ofthe present invention is carried out as described hereinabove.

[0829] (2-2) Screening Method B

[0830] Next, the method of screening a compound that regulates theexpression level of bovine GPR7/8 is explained below.

[0831] The screening method B of the present invention is specifically(i) a method of screening a compound or its salt that increases ordecreases the expression level of bovine GPR7/8, which comprisesassaying the expression level of bovine GPR7/8 or the level of mRNAencoding the bovine GPR7/8 in the case that a cell or tissue capable ofexpressing the bovine GPR7/8 is cultured in the presence or absence of atest compound, and comparing the expression level in each case.

[0832] As the cell or tissue capable of expressing the bovine GPR7/8,there may be used a cell or tissue derived from human or otherwarm-blooded animals (e.g., guinea pigs, rats, mice, chicken, rabbits,swine, sheep, bovine, monkeys, etc.); any cell (e.g., nerve cells,endocrine cells, neuroendocrine cells, glial cells, β cells of pancreas,bone marrow cells, hepatocytes, splenocytes, mesangial cells, epidermiccells, epithelial cells, endothelial cells, fibroblasts, fibrocytes,myocytes, fat cells, immune cells (e.g., macrophages, T cells, B cells,natural killer cells, mast cells, neutrophils, basophils, eosinophils,monocytes, dendritic, cells), megakaryocyte, synovial cells,chondrocytes, bone cells, osteoblasts, osteoclasts, mammary gland cellsor interstitial cells, the corresponding precursor cells, stem cells,cancer cells, etc., of these cells), or any tissue where such a cell ispresent, e.g., brain or any region of the brain (e.g., olfactory bulb,amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus,cerebral cortex, medulla oblongata, cerebellum), spinal cord,hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid,gall-bladder, bone marrow, adrenal gland, skin, muscle, lung,gastrointestinal tract (e.g., large intestine and small intestine),blood vessel, heart, thymus, spleen, submandibular gland, peripheralblood, prostate, testis, ovary, placenta, uterus, bone, cartilage,joint, skeletal muscle, etc., wherein established cell line or primaryculture system may also be used. Transformants transformed by arecombinant vector bearing a DNA encoding the bovine GPR7/8 describedabove may also be used.

[0833] To cultivate the cells capable of expressing the bovine GPR7/8,the method given for cultivating transformants above applies.

[0834] As the test compound, a DNA library may also be used, in additionto the test compounds described above.

[0835] The expression level of bovine GPR7/8 can be determined bypublicly known methods such as immunochemical methods, etc., using anantibody, etc. Alternatively, mRNA encoding the bovine GPR7/8 can bedetermined by publicly known methods including northern hybridization,RT-PCR or TaqMan PCR.

[0836] Comparison of the expression level of mRNA can be made bypublicly known methods or a modification thereof, for example, accordingto the method described in Molecular Cloning, 2nd (J. Sambrook et al.,Cold Spring Harbor Lab. Press, 1989), etc.

[0837] Specifically, the level of mRNA encoding the bovine GPR7/8 isdetermined by contacting RNA extracted from cells according to publiclyknown methods with the DNA encoding the bovine GPR7/8 or a part thereofor the bovine GPR7/8 antisense polynucleotide of the present invention,and assaying the level of mRNA bound to the DNA encoding the bovineGPR7/8 or a part thereof or the antisense polynucleotide of the presentinvention. The level of mRNA bound to the DNA encoding the bovine GPR7/8or a part thereof or the bovine GPR7/8 antisense polynucleotide of thepresent invention can be readily assayed by labeling the DNA encodingthe bovine GPR7/8 or a part thereof or the bovine GPR7/8 antisensepolynucleotide of the present invention with, e.g., a radioisotope, adye, etc. Examples of the radioisotope are ³²P, ³H, etc. Examples of thedye used are fluorescent dyes such as fluorescein, FAM (Biosystems,Inc.), JOE (PE Biosystems, Inc.), TAMRA (PE Biosystems, Inc.), ROX (PEBiosystems, Inc.), Cy5 (Amersham), Cy3 (Amersham), etc.

[0838] The level of mRNA can also be determined by converting RNAextracted from cells into cDNA by a reverse transcriptase, amplifyingthe cDNA by PCR using the DNA encoding the bovine GPR7/8 or a partthereof or the bovine GPR7/8 antisense polynucleotide of the presentinvention as a primer, and assaying the amount of cDNA amplified.

[0839] As described above, the test compound that increases the level ofmRNA encoding the bovine GPR7/8 can be selected as a compound thatincreases the expression level of bovine GPR7/8. Also, the test compoundthat decreases the level of mRNA encoding the bovine GPR7/8 can beselected as a compound that decreases the expression level of bovineGPR7/8.

[0840] The present invention further provides:

[0841] (ii) a method of screening a compound that promotes or inhibits apromoter activity, which comprises assaying the reporter activity in thecase that a transformant transformed by a recombinant DNA ligated with areporter gene downstream the promoter region or enhancer region of agene encoding the bovine GPR7/8 is cultured in the presence or absenceof a test compound, and comparing the activity in each case.

[0842] As the reporter gene, there may be employed, e.g., lacZ(β-galactosidase gene), chloramphenicol acetyltransferase (CAT),luciferase, growth factor, β-glcuronidase, alkaline phosphatase, greenfluorescent protein (GFP), β-lactamase, etc.

[0843] By determining the level of the reporter gene product (e.g.,mRNA, protein) using publicly known methods, the test compound thatincreases the level of the reporter gene product can be selected as thecompound having the activity of regulating (especially promoting) thepromoter or enhancer activity of bovine GPR7/8 of the present invention,i.e., the compound having the activity of increasing the expressionlevel of bovine GPR7/8. To the contrary, the test compound thatdecreases the level of the reporter gene product can be selected as thecompound having the activity of regulating (especially inhibiting) thepromoter or enhancer activity of bovine GPR7/8, i.e., the compoundhaving the activity of decreasing the expression level of bovine GPR7/8.

[0844] As the test compounds, those described above are employed.

[0845] The transformants can be cultivated as given for thetransformants described above.

[0846] Construction of vectors for the reporter genes and assay can beperformed according to publicly known techniques (e.g., MolecularBiotechnology, 13, 29-43, 1999).

[0847] The compounds having the activity of increasing the expressionlevel of bovine GPR7/8 are useful-as safe and low-toxic drugs (e.g.,preventive/therapeutic agents for anorexia, appetite (eating)stimulants, preventive/therapeutic agents for pituitary hormonesecretion disorders [e.g., prolactin secretion disorders (e.g.,hypoovarianism, spermatic underdevelopment, menopausal symptoms,hypothyroidism, etc.)].

[0848] The compounds having the activity of decreasing the expressionlevel of bovine GPR7/8 are useful as safe and low-toxic drugs for theprevention/treatment of obesity (e.g., malignant mastocytosis, exogenousobesity, hyperinsulinar obesity, hyperplasmic obesity, hypophysealadiposity, hypoplasmic obesity, hypothyroid obesity, hypothalamicobesity, symptomatic obesity, infantile obesity, upper body obesity,alimentary obesity, hypogonadal obesity, systemic mastocytosis, simpleobesity, central obesity, etc.), hyperphagia, etc.; as safe andlow-toxic drugs for the prevention/treatment (prolactin productionsuppressing agents) for pituitary tumor, diencephalon tumor, menstrualdisorders, autoimmune disease, prolactinoma, sterility, impotence,amenorrhea, lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-delCastillo syndrome, Forbes-Albright syndrome, lymphoma or Sheehan'ssyndrome, spermatogenesis disorder, etc.; preferably, as safe andlow-toxic drugs for the prevention/treatment of obesity, hyperphagia,etc.

[0849] The compound or its salt, which can be obtained by using thescreening method B or the screening kit of the present invention, is acompound selected from, e.g., peptides, proteins, non-peptide compounds,synthetic compounds, fermentation products, cell extracts, plantextracts, animal tissue extracts, plasma, etc., and is the compound thatpromotes or inhibits the function of the peptide of the presentinvention.

[0850] For salts of the compound, those as described for the peptide ofthe present invention are employed.

[0851] When the compound obtained by the screening method B or screeningkit of the present invention is used as the therapeutic/preventive agentdescribed above, the compound can be prepared into pharmaceuticalpreparations and provided for use, in a similar manner to the aforesaidpharmaceuticals comprising the compound or its salt that alters theexpression level of the peptide of the present invention describedabove.

[0852] (3) Quantification of the Bovine GPR7/8 of the Present Invention

[0853] The antibody of the present invention is capable of specificallyrecognizing the bovine GPR7/8 of the present invention, and can thus beused for quantification of the bovine GPR7/8 of the present invention ina sample fluid, in particular, for quantification by sandwichimmunoassay.

[0854] That is, the present invention provides:

[0855] (i) a method for quantification of the bovine GPR7/8 of thepresent invention in a sample fluid, which comprises competitivelyreacting the bovine GPR7/8 antibody of the present invention with asample fluid and a labeled form of the bovine GPR7/8 of the presentinvention, and measuring a ratio of the labeled bovine GPR7/8 of thepresent invention bound to the antibody; and,

[0856] (ii) a method for quantification of the bovine GPR7/8 of thepresent invention in a sample fluid, which comprises simultaneously orcontinuously reacting the sample fluid with the antibody of the presentinvention and a labeled form of another antibody of the presentinvention immobilized on an insoluble carrier, and measuring theactivity of the labeling agent on the immobilized carrier.

[0857] In the method of quantification (ii) described above, it ispreferred that one antibody is capable of recognizing the N-terminalregion of the bovine GPR7/8 of the present invention, while anotherantibody is capable of recognizing the C-terminal region of the bovineGPR7/8 of the present invention.

[0858] The monoclonal antibody to the bovine GPR7/8 of the presentinvention may be used to quantify the bovine GPR7/8 of the presentinvention, and may further be used to detect the same by means of atissue staining, etc. For these purposes, the antibody molecule per semay be used, or F(ab′)₂, Fab′ or Fab fractions of the antibody moleculemay be used as well.

[0859] The method of quantifying the bovine GPR7/8 of the presentinvention using the bovine GPR7/8 antibody of the present invention isnot particularly limited, and any method may be used so far as itrelates to a method, in which the amount of an antibody, antigen orantibody-antigen complex can be detected by a chemical or a physicalmeans, depending on or corresponding to the amount of antigen (e.g., theamount of the peptide) in a sample fluid to be assayed, and thencalculated using a standard curve prepared by a standard solutioncontaining the known amount of antigen. Advantageously used are, forexample, nephrometry, competitive method, immunometric method andsandwich method; in terms of sensitivity and specificity, the sandwichmethod, which will be described later, is particularly preferred.

[0860] Examples of labeling agents, which are employed for the assaymethod using the same, are radioisotopes, enzymes, fluorescentsubstances, luminescent substances, etc. Examples of radioisotopes are[¹²⁵I], [¹³¹H], [³H], [¹⁴C], etc. Preferred examples of enzymes arethose that are stable and have a high specific activity, which includeβ-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malatedehydrogenase, etc. Examples of fluorescent substances arefluorescamine, fluorescein isothiocyanate, etc. Examples of luminescentsubstances are luminol, a luminol derivative, luciferin, lucigenin, etc.Furthermore, a biotin-avidin system may be used as well for binding anantibody or antigen to a labeling agent.

[0861] In the immobilization of antigens or antibodies, physicaladsorption may be used. Alternatively, chemical binding that isconventionally used for immobilization of proteins, enzymes, etc. may beused as well. Examples of the carrier include insoluble polysaccharidessuch as ag arose, dextran, cellulose, etc.; synthetic resins such aspolystyrene, polyacrylamide, silicone, etc.; or glass; and the like.

[0862] In the sandwich method, a sample fluid is reacted with animmobilized form of the bovine GPR7/8 monoclonal antibody of the presentinvention (primary reaction), then reacted with a labeled form of thebovine GPR7/8 monoclonal antibody of the present invention (secondaryreaction) and the activity of the labeling agent on the insolublecarrier is assayed; thus, the amount of the bovine GPR7/8 of the presentinvention in a sample fluid can be determined. The primary and secondaryreactions may be carried out in a reversed order, simultaneously orsequentially with intervals. The type of the labeling agent and themethod of immobilization may be the same as those described hereinabove.In the immunoassay by the sandwich method, it is not always necessarythat the antibody used for the labeled antibody and for the solid phaseshould be one type or one species but a mixture of two or moreantibodies may also be used for the purpose of improving the assaysensitivity, etc.

[0863] In the method of assaying the bovine GPR7/8 of the presentinvention by the sandwich method according to the present invention, thebovine GPR7/8 monoclonal antibodies of the present invention used forthe primary and the secondary reactions are preferably antibodies, whichbinding sites to the bovine GPR7/8 of the present invention aredifferent from each other. Thus, the antibodies used in the primary andsecondary reactions are those wherein, when the antibody used in thesecondary reaction recognizes the C-terminal region of the bovine GPR7/8of the present invention, the antibody recognizing the site other thanthe C-terminal regions, e.g., recognizing the N-terminal region, ispreferably used in the primary reaction.

[0864] The bovine GPR7/8 antibody of the present invention may be usedin an assay system other than the sandwich method, such as thecompetitive method, the immunometric method or the nephrometry.

[0865] In the competitive method, an antigen in a sample fluid and alabeled antigen are competitively reacted with an antibody, then anunreacted labeled antigen (F) and a labeled antigen bound to theantibody (B) are separated (i.e., B/F separation) and the labeled amountof either B or F is measured to determine the amount of the antigen inthe sample fluid. In the reactions for such a method, there are a liquidphase method in which a soluble antibody is used as the antibody and theB/F separation is effected by polyethylene glycol, while a secondantibody to the antibody is used, and a solid phase method in which animmobilized antibody is used as the first antibody or a soluble antibodyis used as the first antibody, while an immobilized antibody is used asthe second antibody.

[0866] In the immunometric method, an antigen in a sample fluid and animmobilized antigen are competitively reacted with a given amount of alabeled antibody followed by separating the solid phase from the liquidphase; or an antigen in a sample fluid and an excess amount of labeledantibody are reacted, then an immobilized antigen is added to bind anunreacted labeled antibody to the solid phase and the solid phase isseparated from the liquid phase. Thereafter, the labeled amount of anyof the phases is measured to determine the antigen amount in the samplefluid.

[0867] In the nephrometry, the amount of insoluble sediment, which isproduced as a result of the antigen-antibody reaction in a gel or in asolution, is measured. Even when the amount of an antigen in a samplefluid is small and only a small amount of the sediment is obtained, alaser nephrometry utilizing laser scattering can be suitably used.

[0868] In applying each of those immunoassays to the assay method of thepresent invention, any special conditions, operations, etc. are notrequired. The assay system for the bovine GPR7/8 of the presentinvention may be constructed in addition to conditions or operationsconventionally used for each of the methods, taking technicalconsideration by one skilled in the art into account. For the details ofsuch conventional technical means, a variety of reviews, referencebooks, etc. may be referred to:

[0869] for example, Hiroshi Irie (ed.): “Radioimmunoassay” (published byKodansha, 1974); Hiroshi Irie (ed:): “Radioimmunoassay; Second Series”(published by Kodansha, 1979); Eiji Ishikawa, et al. (ed.): “EnzymeImmunoassay” (published by Igaku Shoin, 1978); Eiji Ishikawa, et al.(ed.): “Enzyme lhmunoassay” (Second Edition) (published by Igaku Shoin,1982); Eiji Ishikawa, et al. (ed.): “Enzyme Immunoassay” (Third Edition)(published by Igaku Shoin, 1987); “Methods in Enzymology” Vol. 70(Immunochemical Techniques (Part A)); ibid., Vol. 73 (ImmunochemicalTechniques (Part B)); ibid., Vol. 74 (Immunochemical Techniques (PartC)); ibid., Vol. 84 (Immunochemical Techniques (Part. D: SelectedImmunoassays)); ibid., Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies)) (all published by Academic Press); etc.

[0870] As described above, the bovine GPR7/8 of the present inventioncan be quantified with high sensitivity, using the bovine GPR7/8antibody of the present invention.

[0871] Furthermore, when a reduced level of the bovine GPR7/8 of thepresent invention is detected by quantifying a level of the bovineGPR7/8 of the present invention using, the bovine GPR7/8 antibody of thepresent invention, it can be diagnosed that one suffers from, e.g.,anorexia, hypertension, autoimmune disease, heart failure, cataract,glaucoma, acute bacterial meningitis, acute myocardial infarction, acutepancreatitis, acute viral encephalitis, adult respiratory distresssyndrome, alcoholic hepatitis, Alzheimer's disease, asthma,arteriosclerosis, atopic dermatitis, bacterial pneumonia, bladdercancer, fracture, breast cancer, bulimia, polyphagia, burn healing,uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially, anorexia or the like) etc.; or itis highly likely for one to suffer from these disease in the future.

[0872] When an increased level of the bovine GPR7/8 of the presentinvention is detected, it can be diagnosed that one suffers from, e.g.,obesity (e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma, Sheehan's syndrome,spermatogenesis disorder (especially, obesity, or the like), etc.; or itis highly likely for one to suffer from these disease in the future.

[0873] The bovine GPR7/8 antibody of the present invention may also beemployed to detect the bovine GPR7/8 of the present invention present ina sample fluid such as body fluids, tissues, etc. The antibody mayfurther be used for the preparation of an antibody column to purify thebovine GPR7/8 of the present invention, detect the bovine GPR7/8, of thepresent invention in each fraction upon purification, analysis of thebehavior of the bovine GPR7/8 of the present invention in the cellsunder investigation.

[0874] (4) Gene Diagnostic Agent

[0875] By using the bovine GPR7/8 DNA of the present invention, e.g., asa probe, abnormality (gene abnormality) of the DNA or mRNA encoding thebovine GPR7/8 of the present invention in human or other warm-bloodedanimals (e.g., rats, mice, guinea pigs, rabbits, chicken, sheep, swine,bovine, horses, cats, dogs, monkeys, etc.) can be detected. Thus, thebovine GPR7/8 DNA of the present invention is useful as a genediagnostic agent for the damage to the DNA or mRNA, mutation, adecreased expression or an increased expression, or overexpression ofthe DNA or mRNA.

[0876] The gene diagnosis described above using the bovine GPR7/8 DNA ofthe present invention can be performed by, for example, the publiclyknown northern hybridization assay, or the PCR-SSCP assay (Genomics, 5,874-879 (1989); Proceedings of the National Academy of Sciences of theUnited States of America, 86, 2766-2770 (1989)), etc.

[0877] When a decreased expression of mRNA is detected, e.g., bynorthern hybridization, it can be diagnosed that one is likely to sufferfrom, for example, anorexia, hypertension, autoimmune disease, heartfailure, cataract, glaucoma, acute bacterial meningitis, acutemyocardial infarction, acute pancreatitis, acute viral encephalitis,adult respiratory distress syndrome, alcoholic hepatitis, Alzheimer'sdisease, asthma, arteriosclerosis, atopic dermatitis, bacterialpneumonia, bladder cancer, fracture, breast cancer, bulimia, polyphagia,burn healing, uterine cervical cancer, chronic lymphocytic leukemia,chronic myelogenous leukemia, chronic pancreatitis, liver cirrhosis,cancer of the colon and rectum (colon cancer/rectal cancer), Crohn'sdisease, dementia, diabetic complications, diabetic nephropathy,diabetic neuropathy, diabetic retinopathy, gastritis, Helicobacterpylori bacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation; arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially anorexia or the like) etc.; or itis highly likely for one to suffer from diseases in the future.

[0878] When overexpression of mRNA is detected by northernhybridization, it can be diagnosed that one is likely to suffer from,for example, obesity (e.g., malignant mastocytosis, exogenous obesity,hyperinsulinar obesity, hyperplasmic obesity, hypophyseal adiposity,hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity,symptomatic obesity, infantile obesity, upper body obesity, alimentaryobesity, hypogonadal obesity, systemic mastocytosis, simple obesity,central obesity, etc.), hyperphagia, pituitary tumor, diencephalontumor, menstrual disorder, autoimmune disease, prolactinoma, sterility,impotence, amenorrhea, lactorrhea, acromegaly, Chiari-Frommel syndrome,Argonz-del Castillo syndrome, Forbes-Albright syndrome, lymphoma orSheehan's syndrome, spermatogenesis disorder, etc. (especially, obesityor the like); or it is highly likely for one to suffer from diseases inthe future.

[0879] (5) Pharmaceutical Composition comprising Bovine GPR7/8 AntisenseDNA

[0880] The antisense DNA that binds complementarily to the bovine GPR7/8DNA of the present invention to inhibit expression of the DNA can beused as preventive/therapeutic agents, for example, for obesity (e.g.,malignant mastocytosis, exogenous obesity, hyperinsulinar obesity,hyperplasmic obesity, hypophyseal adiposity, hypoplasmic obesity,hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc. (especially, obesity or the like), etc.

[0881] For example, when the antisense DNA is used, the antisense DNAmay be administered directly, or the DNA is inserted into an appropriatevector such as retrovirus vector, adenovirus vector,adenovirus-associated virus vector, etc. and then administered in aconventional manner. The antisense DNA may also be administered as anintact DNA, or prepared into pharmaceutical preparations together with aphysiologically acceptable carrier such as an adjuvant to assist itsuptake and administered by gene gun or through a catheter such as acatheter with a hydrogel.

[0882] In addition, the antisense DNA may also be employed as anoligonucleotide probe for diagnosis to examine the presence of thebovine GPR7/8 DNA of the present invention in tissues or cells andstates of its expression.

[0883] (6) Pharmaceutical Composition Comprising the Bovine GPR7/8Antibody of the Present Invention

[0884] The bovine GPR7/8 antibody of the present invention having aneffect to neutralize the activity of the bovine GPR7/8 of the presentinvention can be used as drugs for the prevention/treatment of, forexample, obesity (e.g., malignant mastocytosis, exogenous obesity,hyperinsulinar obesity, hyperplasmic obesity, hypophyseal adiposity,hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity,symptomatic obesity, infantile obesity, upper body obesity, alimentaryobesity, hypogonadal obesity, systemic mastocytosis, simple obesity,central obesity, etc.), hyperphagia, pituitary tumor, diencephalontumor, menstrual disorder, autoimmune disease, prolactinoma, sterility,impotence, amenorrhea, lactorrhea, acromegaly, Chiari-Frommel syndrome,Argonz-del Castillo syndrome, Forbes-Albright syndrome, lymphoma orSheehan's syndrome, spermatogenesis disorder, etc. (especially, obesity,or the like), etc.

[0885] The therapeutic/preventive agent comprising the bovine GPR7/8antibody of the present invention can be manufactured in a similarmanner to the pharmaceuticals comprising the antibody to the peptide ofthe present invention described above and provided for use.

[0886] (7) Bovine GPR7/8 DNA Transgenic Animal

[0887] The present invention provides a non-human mammal bearing anexogenous DNA encoding the bovine GPR7/8 of the present invention(hereinafter merely referred to as the exogenous bovine GPR7/8 DNA ofthe present invention) or its variant DNA (sometimes simply referred toas the exogenous variant bovine GPR7/8 DNA of the present invention).

[0888] Thus, the present invention provides:

[0889] (i) a non-human mammal bearing the exogenous bovine GPR7/8 DNA orits variant DNA;

[0890] (ii) the mammal according to (i), wherein the non-human mammal isa rodent;

[0891] (iii) the mammal according to (ii), wherein the rodent is mouseor rat; and,

[0892] (iv) a recombinant vector bearing the exogenous bovine GPR7/8 DNAof the present invention or its variant DNA and capable of expressing ina mammal.

[0893] The bovine GPR7/8 DNA transgenic animals of the present inventioncan be prepared in a manner similar to the DNA transgenic animals of thepresent invention described above.

[0894] In a non-human mammal bearing the normal bovine GPR7/8 DNA of thepresent invention, the normal bovine GPR7/8 DNA of the present inventionis expressed to a high level, and may eventually develop thehyperfunction of the bovine GPR7/8 of the present invention by promotingthe function of endogenous normal bovine GPR7/8 DNA. Therefore, theanimal can be utilized as a pathologic model animal for such a disease.Specifically, using the normal bovine GPR7/8 DNA transgenic animal ofthe present invention, it is possible to elucidate the mechanism of thehyperfunction of the bovine GPR7/8 of the present invention and thepathological mechanism of the disease associated with the bovine GPR7/8of the present invention and to determine how to treat the disease.

[0895] Furthermore, since a mammal wherein the exogenous normal bovineGPR7/8 DNA of the present invention is transfected exhibits anincreasing symptom of the bovine GPR7/8 of the present inventionlibrated, the animal is usable for screening therapeutic agents for thedisease associated with the bovine GPR7/8 of the present invention.

[0896] On the other hand, in a non-human mammal bearing the abnormalbovine GPR7/8 DNA of the present invention, the abnormal bovine GPR7/8DNA of the present invention is expressed at a high level, the animalmay be the function inactivation type inadaptability of the bovineGPR7/8 of the present invention by inhibiting the function of theendogenous normal bovine GPR7/8 DNA and can be utilized as its diseasemodel animal. For example, using the abnormal bovine GPR7/8DNA-transgenic animal of the present invention, it is possible toelucidate the mechanism of inadaptability of the bovine GPR7/8 of thepresent invention and to perform to study a method for treatment of thisdisease.

[0897] More specifically, the transgenic animal of the present inventionexpressing the abnormal bovine GPR7/8 DNA of the present invention to ahigh level is also expected to serve as an experimental model for theelucidation of the mechanism of the functional inhibition (dominantnegative effect) of normal bovine GPR7/8 by the abnormal bovine GPR7/8of the present invention in the function inactive type inadaptability ofthe bovine GPR7/8 of the present invention.

[0898] The abnormal exogenous bovine GPR7/8 DNA transgenic mammal of thepresent invention is also expected to serve for screening a candidatedrug for the treatment of the function inactive type inadaptability ofthe bovine GPR7/8 of the present invention, since the bovine GPR7/8 ofthe present invention is increased in such an animal in its free form.

[0899] Other potential applications of two kinds of the bovine GPR7/8DNA transgenic animals described above include:

[0900] (1) use as a cell source for tissue culture;

[0901] (2) elucidation of the relation to a peptide that is specificallyexpressed or activated by the bovine GPR7/8 of the present invention, bydirect analysis of DNA or RNA in tissues of the bovine GPR7/8 DNAtransgenic animal of the present invention or by analysis of a peptidetissue expressed by the DNA;

[0902] (3) research in the function of cells derived from tissues thatare cultured usually only with difficulty, using cells of tissue bearingthe DNA cultured by a standard tissue culture technique;

[0903] (4) screening of a drug that enhances the functions of cellsusing the cells described in (3) above; and, (5) isolation andpurification of the variant bovine GPR7/8 of the present invention andpreparation of an antibody thereto.

[0904] Furthermore, clinical conditions of a disease associated wit thebovine GPR7/8 of the present invention, including the function inactivetype inadaptability of the bovine GPR7/8 of the present invention can bedetermined using the bovine GPR7/8 DNA transgenic animal of the presentinvention. Also, pathological findings on each organ in a disease modelassociated with the bovine GPR7/8 of the present invention can beobtained in more detail, leading to the development of a new method fortreatment as well as the research and therapy of any secondary diseasesassociated with the disease.

[0905] It is also possible to obtain a free DNA-transfected cell bywithdrawing each organ from the bovine GPR7/8 DNA transgenic animal ofthe present invention, mincing the organ and degrading with a proteinasesuch as trypsin, etc., followed by establishing the line of culturing orcultured cells. Furthermore, the transgenic animal can serve asidentification of cells capable of producing the bovine GPR7/8 of thepresent invention, and as studies on association with apoptosis,differentiation or propagation or on the mechanism of signaltransduction in these properties to inspect any abnormality therein.Thus, the transgenic animal can provide an effective research materialfor the bovine GPR7/8 of the present invention and for elucidating thefunction and effect thereof.

[0906] To develop a therapeutic drug for the treatment of diseasesassociated with the bovine GPR7/8 of the present invention, includingthe function inactive type inadaptability of the bovine GPR7/8 of thepresent invention, using the bovine GPR7/8 DNA transgenic animal of thepresent invention, an effective and rapid method for screening can beprovided by using the method for inspection and the method forquantification, etc. described above. It is also possible to investigateand develop a method for DNA therapy for the treatment of diseasesassociated with the bovine GPR7/8 of the present invention, using thebovine GPR7/8 DNA transgenic animal of the present invention or a vectorcapable of expressing the exogenous bovine GPR7/8 DNA of the presentinvention.

[0907] (8) Knockout Animal

[0908] The present invention provides a non-human mammal embryonic stemcell bearing the bovine GPR7/8 DNA of the present invention inactivatedand a non-human mammal deficient in expressing the bovine GPR7/8 DNA ofthe present invention.

[0909] Thus, the present invention provides:

[0910] (i) a non-human embryonic stem cell in which the bovine GPR7/8DNA of the present invention is inactivated;

[0911] (ii) an embryonic stem cell according to (i), wherein the DNA isinactivated by introducing a reporter gene (e.g., β-galactosidase genederived from Escherichia coli);

[0912] (iii) an embryonic stem cell according to (i), which is resistantto neomycin;

[0913] (iv) an embryonic stem cell according to (i), wherein thenon-human mammal is a rodent;

[0914] (v) an embryonic stem cell according to (iv), wherein the rodentis mouse;

[0915] (vi) a non-human mammal deficient in expressing the bovine GPR7/8DNA of the present invention, wherein the DNA is inactivated;

[0916] (vii) a non-human mammal according to (vi), wherein the DNA isinactivated by inserting a reporter gene (e.g., β-galactosidase derivedfrom Escherichia coli) therein and the reporter gene is capable of beingexpressed under control of a promoter for the DNA of the presentinvention;

[0917] (viii) a non-human mammal according to (vi), which is a rodent;

[0918] (ix) a non-human mammal according to (viii), wherein the rodentis mouse; and,

[0919] (x) a method of screening a compound or its salt that promotes orinhibits the promoter activity for the bovine GPR7/8 DNA of the presentinvention, which comprises administering a test compound to the mammalof (vii) and detecting expression of the reporter gene.

[0920] The non-human mammal embryonic stem cell wherein the bovineGPR7/8 DNA of the present invention is inactivated, and the non-humanmammal deficient in expressing the bovine GPR7/8 DNA of the presentinvention wherein the DNA is inactivated can be prepared as in thenon-human mammal embryonic stem cell of the present invention and thenon-human mammal deficient in expressing the DNA of the presentinvention described above.

[0921] The non-human mammal embryonic stem cell, in which the bovineGPR7/8 DNA of the present invention is inactivated, is very useful forpreparing a non-human mammal deficient in expressing the bovine GPR7/8DNA of the present invention.

[0922] Since the non-human mammal deficient in expressing the bovineGPR7/8 DNA of the present invention lacks various biological activitiesderived from the bovine GPR7/8 of the present invention, such an animalcan be a disease model suspected of inactivated biological activities ofthe bovine GPR7/8 of the present invention and thus, offers an effectivestudy to investigate causes for and therapy for these diseases.

[0923] (8a) Method of Screening Compounds having Therapeutic/PreventiveEffects on Diseases caused by Deficiency, Damages, etc. of the BovineGPR7/8 DNA of the Present Invention

[0924] The non-human mammal deficient in expressing the bovine GPR7/8DNA of the present invention can be employed for screening of compoundshaving therapeutic/prophylactic effects on diseases caused bydeficiency, damages, etc. of the bovine GPR7/8 DNA of the presentinvention.

[0925] That is, the present invention provides a method for screening ofa compound or its salt having therapeutic/preventive effects on diseasescaused by deficiency, damages, etc. of the bovine GPR7/8 DNA of thepresent invention, which comprises administering a test compound to thenon-human mammal deficient in expressing the bovine GPR7/8 DNA of thepresent invention and observing/measuring a change occurred in theanimal.

[0926] As the non-human mammal deficient in expressing the bovineGPR7/8-DNA of the present invention which can be employed for thescreening method, the same examples as given hereinabove apply.

[0927] Examples of the test compounds include peptides, proteins, nonpeptide compounds, synthetic compounds, fermentation products, cellextracts, vegetable extracts, animal tissue extracts, blood plasma, etc.These compounds may be novel compounds or publicly known compounds.

[0928] Specifically, the non-human mammal deficient in expressing thebovine GPR7/8 DNA of the present invention is treated with a testcompound, comparison is made with an intact animal for control and achange in each organ, tissue, disease conditions, etc. of the animal isused as an indicator to assess the therapeutic/prophylactic effects ofthe test compound.

[0929] For treating an animal to be test with a test compound, forexample, oral administration, intravenous injection, etc. are appliedand the treatment is appropriately selected depending upon conditions ofthe test animal, properties of the test compound, etc. Furthermore, adose of test compound to be administered can be appropriately chosendepending on method for administration, nature of the test compound,etc.

[0930] In screening compounds having the therapeutic/preventive effecton, e.g., anorexia, hypertension, autoimmune disease, heart failure,cataract, glaucoma, acute bacterial meningitis, acute myocardialinfarction, acute pancreatitis, acute viral encephalitis, adultrespiratory distress syndrome, alcoholic hepatitis, Alzheimer's disease,asthma, arteriosclerosis, atopic dermatitis, bacterial pneumonia,bladder cancer, fracture, breast cancer, bulimia, polyphagia, burnhealing, uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease, etc. (especially, anorexia, or the like), thenon-human mammal deficient in expressing the bovine GPR7/8 DNA of thepresent invention is subjected to a sugar loading treatment, a testcompound is administered before or after the sugar loading treatmentand, blood sugar level, body weight change, etc. of the animal ismeasured with passage of time.

[0931] In the screening method described above, when a test compound isadministered to a test animal and found to reduce the blood sugar levelof the animal to at least about 10%, preferably at least about 30% andmore preferably at least about 50%, the test compound can be selected tobe a compound having a therapeutic/preventive effect on the diseasesabove.

[0932] The compound obtained using the screening method above is acompound selected from the test compounds described above and exhibits atherapeutic/preventive effect on the diseases caused by deficiencies,damages, etc. of the bovine GPR7/8 of the present invention. Therefore,the compound can be employed as a safe and low toxic drug for thetreatment and prevention of these diseases. Furthermore, compoundsderived from such a compound obtained by the screening described abovecan be similarly employed.

[0933] The compound obtained by the screening method above may be in theform of salts. As such salts, there may be used salts withphysiologically acceptable acids (e.g., inorganic acids, organic acids,etc.) or bases (e.g., alkali metal salts, etc.), preferably in the formof physiologically acceptable acid addition salts. Examples of suchsalts are salts with inorganic acids (e.g., hydrochloric acid,phosphoric acid, hydrobromic acid, sulfuric acid, etc.), salts withorganic acids (e.g., acetic acid, formic acid, propionic acid, fumaricacid, maleic acid, succinic acid, tartaric acid, citric acid, malicacid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonicacid, etc.) and the like.

[0934] A pharmaceutical composition comprising the compound or its salt,obtained by the above screening method, may be manufactured in a mannersimilar to the method for preparing the pharmaceutical compositioncomprising the peptide of the present invention described hereinabove.

[0935] Since the pharmaceutical composition thus obtained is safe andlow toxic, it can be administered to human or mammals (e.g., rats, mice,guinea pigs, rabbits, sheep, swine, bovine, horses, cats, dogs, monkeys,etc.).

[0936] A dose of the compound or its salt to be administered variesdepending upon particular disease, subject to be administered, route ofadministration, etc., and in oral administration to an adult patientwith anorexia (as 60 kg body weight), the compound is administeredgenerally in a dose of approximately 0.1 to. 100 mg, preferablyapproximately 1.0 to 50 mg, more preferably approximately 1.0 to 20 mgper day. For parenteral administration to an adult patient with anorexia(as 60 kg body weight), it is advantageous to administer the compoundintravenously in the form of an injectable preparation in a dose ofapproximately 0.01 to 30 mg, preferably approximately 0.1 to 20 mg, morepreferably approximately 0.1 to 10 mg per day, though the single dosagevaries depending upon particular subject, particular disease, etc. Forother animals, the compound can be administered in the correspondingdose with converting it into that for the 60 kg body weight.

[0937] (8b) Method of Screening a Compound that Promotes or Inhibits theActivity of a Promoter to the Bovine GPR7/8 DNA of the Present Invention

[0938] The present invention provides a method of screening a compoundor its salt that promotes or inhibits the activity of a promoter to thebovine GPR7/8 DNA of the present invention, which comprisesadministering a test compound to a non-human mammal deficient inexpressing the bovine GPR7/8 DNA of the present invention and detectingexpression of the reporter gene.

[0939] In the screening method described above, the non-human mammaldeficient in expressing the bovine GPR7/8 DNA of the present inventionis selected from the aforesaid non-human mammal deficient in expressingthe bovine GPR7/8 DNA of the present invention, as an animal in whichthe bovine GPR7/8 DNA of the present invention is inactivated byintroducing a reporter gene and the reporter gene is expressed undercontrol of a promoter to the bovine GPR7/8 DNA of the present invention.

[0940] The same examples of the test compound apply to those givenabove.

[0941] As the reporter gene, the same specific examples apply.Preferably employed are β-galactosidase (lacZ), soluble alkalinephosphatase gene, luciferase gene and the like.

[0942] Since the reporter gene is present under control of a promoter tothe bovine GPR7/8 DNA of the present invention in the non-human mammaldeficient in expressing the bovine GPR7/8 DNA of the present inventionwherein the bovine GPR7/8 DNA of the present invention is substitutedwith the reporter gene, the activity of the promoter can be detected bytracing expression of a substance encoded by the reporter gene.

[0943] For example, when a part of the DNA region encoding the bovineGPR7/8 of the present invention is substituted with, e.g.,β-galactosidase gene (lacZ) derived from Escherichia coli,β-galactosidase is expressed in a tissue where the bovine GPR7/8 of thepresent invention should originally be expressed, instead of the bovineGPR7/8 of the present invention. Thus, the state of expression conditionof the bovine GPR7/8 of the present invention can be readily observed invivo of an animal by staining with a reagent, e.g.,5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal) which issubstrate for β-galactosidase. Specifically, a mouse deficient in thebovine GPR7/8 of the present invention, or its tissue slice section isfixed with glutaraldehyde, etc. After washing with phosphate bufferedsaline (PBS), the system is reacted with a staining solution containingX-gal at room temperature or about 37° C. for approximately 30 minutesto an hour. After the β-galactosidase reaction is terminated by washingthe tissue preparation with 1 mM EDTA/PBS solution, the color formed isobserved. Alternatively, mRNA encoding lacZ may be detected in aconventional manner.

[0944] The compound or salts thereof obtained using the aforesaidscreening method are compounds that are selected from the test compoundsdescribed above and the compounds that promote or inhibit the activityof a promoter to the bovine GPR7/8 DNA of the present invention.

[0945] The compound obtained by the screening method above may formsalts. As salts of the compound, there may be used salts withphysiologically acceptable acids (e.g., inorganic acids, etc.) or bases(e.g., organic acids, etc.), and especially preferred arephysiologically acceptable acid addition salts. Examples of such saltsare salts with inorganic acids (e.g., hydrochloric acid, phosphoricacid, hydrobromic acid, sulfuric acid, etc.), salts with organic acids(e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleicacid, succinic acid, tartaric acid, citric acid, malic acid, oxalicacid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.)and the like.

[0946] The compound or its salt that promotes the promoter activity tothe bovine GPR7/8 DNA of the present invention can promote expression ofthe bovine GPR7/8 of the present invention thereby to promote thefunction of the bovine GPR7/8. Thus, these compounds are useful as safeand low-toxic drugs for the treatment/prevention of diseases, e.g.,anorexia, hypertension, autoimmune disease, heart failure, cataract,glaucoma, acute bacterial meningitis, acute myocardial infarction, acutepancreatitis, acute viral encephalitis, adult respiratory distresssyndrome, alcoholic hepatitis, Alzheimer's disease, asthma,arteriosclerosis, atopic dermatitis, bacterial pneumonia, bladdercancer, fracture, breast cancer, bulimia, polyphagia, burn healing,uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease, etc. (especially, anorexia or the like) orthe like (especially, appetite (eating) stimulant).

[0947] The compound or its salt that inhibits the activity of a promoterto the bovine GPR7/8 DNA of the present invention can inhibit expressionof the bovine GPR7/8 of the present invention thereby to inhibit thefunction of the bovine GPR7/8. Thus, these compounds are useful asdrugs, including preventive/therapeutic drugs (prolactin productioninhibitors) for diseases, for example, obesity (e.g., malignantmastocytosis, exogenous obesity, hyperinsulinar obesity, hyperplasmicobesity, hypophyseal adiposity, hypoplasmic obesity, hypothyroidobesity, hypothalamic obesity, symptomatic obesity, infantile obesity,upper body obesity, alimentary obesity, hypogonadal obesity, systemicmastocytosis, simple obesity, central obesity, etc.), hyperphagia,pituitary tumor, diencephalon tumor, menstrual disorder, autoimmunedisease, prolactinoma, sterility, impotence, amenorrhea, lactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorders, etc.; preferably as preventive/therapeuticagents for obesity, hyperphagia, etc.

[0948] Furthermore, compounds derived from the compounds obtained by thescreening described above may be likewise used.

[0949] The pharmaceuticals comprising the compound or its salt obtainedby the screening method may be manufactured as in the aforesaidpharmaceuticals comprising the peptide of the present invention or itssalt.

[0950] Since the pharmaceutical preparation thus obtained is safe andlow toxic; it can be administered to human or mammals (e.g., rats, mice,guinea pigs, rabbits, sheep, swine, bovine, horses, cats, dogs, monkeys,etc.).

[0951] A dose of the compound or its salt to be administered variesdepending upon target disease, subject to be administered, route ofadministration, etc.; when the compound that promotes the promoteractivity to the bovine GPR7/8 DNA of the present invention is orallyadministered to an adult patient, e.g., with anorexia (as 60 kg bodyweight), the compound is administered generally in a dose ofapproximately 0.1 to 100 mg, preferably approximately 1.0 to 50 mg, morepreferably approximately 1.0 to 20 mg per day. In parenteraladministration, a single dose of the compound varies depending uponsubject to be administered, target disease, etc. When the compound thatpromotes the promoter activity to the bovine GPR7/8 DNA of the presentinvention is administered to an adult patient with, e.g., anorexia (as60 kg body weight) in the form of an injectable preparation, it isadvantageous to administer the compound intravenously in a dose ofapproximately 0.01 to 30 mg, preferably approximately 0.1 to 20 mg, morepreferably approximately 0.1 to 10 mg per day. For other animals, thecompound can be administered in the corresponding dose with convertingit into that for the 60 kg body weight.

[0952] On the other hand, when a compound that inhibits the promoteractivity to the bovine GPR7/8 DNA of the present invention is orallyadministered, the compound is orally administered to an adult patientwith obesity (as 60 kg body weight) generally in a dose of approximately0.1 to 100 mg, preferably approximately 1.0 to 50 mg, more preferablyapproximately 1.0 to 20 mg per day. In parenteral administration, asingle dose of the compound varies depending upon subject to beadministered, target disease, etc. For example, when the compound thatinhibits the promoter activity to the bovine GPR7/8 DNA of the presentinvention is administered to an adult patient with obesity (as 60 kgbody weight) in the form of an injectable preparation, it isadvantageous to administer the compound intravenously in a dose ofapproximately 0.01 to 30 mg, preferably approximately 0.1 to 20 mg, morepreferably approximately 0.1 to 10 mg per day. For other animals, thecompound can be administered in the corresponding dose with convertingit into that for the 60 kg body weight.

[0953] As described above, the non-human mammal deficient in expressingthe bovine GPR7/8 DNA of the present invention is extremely useful forscreening a compound or its salt that promotes or inhibits the activityof a promoter to the bovine GPR7/8 DNA of the present invention, and canthus greatly contribute to investigations of causes for various diseasescaused by failure to express the bovine GPR7/8 DNA of the presentinvention or to development of preventive/therapeutic agents for thesediseases.

[0954] Moreover, when a so-called transgenic animal (gene-transfectedanimal) is prepared by using the bovine GPR7/8 DNA containing thepromoter region of the bovine GPR7/8 of the present invention, ligatinggenes encoding various proteins downstream the same and injecting thegenes into animal oocyte, the peptide can be specifically synthesized bythe animal so that it becomes possible to investigate the activity invivo. Furthermore, when an appropriate reporter gene is ligated to thepromoter region described above to establish a cell line so as toexpress the gene, such can be used as a survey system of low molecularweight compounds that specifically promotes or suppresses the ability ofproducing the bovine GPR7/8 itself of the present invention in vivo.

[0955] In the specification and drawings, the codes of bases and aminoacids are shown by abbreviations and in this case, they are denoted inaccordance with the IUPAC-IUB Commission on Biochemical Nomenclature orby the common codes in the art, examples of which are shown below. Foramino acids that may have the optical isomer, L form is presented unlessotherwise indicated.

[0956] DNA: deoxyribonucleic acid

[0957] cDNA: complementary deoxyribonucleic acid

[0958] A: adenine

[0959] T: thymine

[0960] G: guanine

[0961] C: cytosine

[0962] I: inosine

[0963] R: adenine (A) or guanine (G)

[0964] Y: thymine (T) or cytosine (C)

[0965] M: adenine (A) or cytosine (C)

[0966] K: guanine (G) or thymine (T)

[0967] S: guanine (G) or cytosine (C)

[0968] W: adenine (A) or thymine (T)

[0969] B: guanine (G), guanine (G) or thymine (T)

[0970] D: adenine (A), guanine (G) or thymine (T)

[0971] V: adenine (A), guanine (G) or cytosine (C)

[0972] N: adenine (A), guanine (G), cytosine (C) or thymine (T), orunknown or other base

[0973] RNA: ribonucleic acid

[0974] mRNA: messenger ribonucleic acid

[0975] dATP: deoxyadenosine triphosphate

[0976] dTTP: deoxythymidine triphosphate

[0977] dGTP: deoxyguanosine triphosphate

[0978] dCTP: deoxycytidine triphosphate

[0979] ATP: adenosine triphosphate

[0980] EDTA: ethylenediaminetetraacetic acid

[0981] SDS: sodium dodecyl sulfate

[0982] BHA: benzhydrylamine

[0983] pMBHA: p-methyobenzhydrylamine

[0984] Tos: p-toluenesulfonyl.

[0985] Bzl: benzyl

[0986] Bom: benzyloxymethyl

[0987] Boc: t-butyloxycarbonyl

[0988] DCM: dichloromethane

[0989] HOBt: 1-hydroxybenztriazole

[0990] DCC: N,N′-dicyclohexylcarbodiimide

[0991] TFA: trifluoroacetic acid

[0992] DIEA: diisopropylethylamine

[0993] Gly or G: glycine

[0994] Ala or A: alanine

[0995] Val or V: valine

[0996] Leu or L: leucine

[0997] Ile or I: isoleucine

[0998] Ser or S: serine

[0999] Thr or T: threonine

[1000] Cys or C: cysteine

[1001] Met or M: methionine

[1002] Glu or E: glutamic acid

[1003] Asp or D: aspartic acid

[1004] Lys or K: lysine

[1005] Arg or R: arginine

[1006] His or H: histidine

[1007] Phe or F: phenylalanine

[1008] Tyr or Y: tyrosine

[1009] Trp or W: tryptophan

[1010] Pro or P: proline

[1011] Asn or N: asparagine

[1012] Gln or Q: glutamine

[1013] pGlu: pyroglutamic acid

[1014] Tyr (I): 3-iodotyrosine

[1015] DMF: N,N-dimethylformamide

[1016] Fmoc: N-9-fluorenylmethoxycarbonyl

[1017] Trt: trityl

[1018] Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

[1019] Clt: 2-chlorotrityl

[1020] Bu^(t): t-butyl

[1021] Met (O): methionine sulfoxide

[1022] The sequence identification numbers in the sequence listing ofthe specification indicates the following sequences, respectively.

[1023] [SEQ ID NO:1]

[1024] This shows the amino acid sequence of human GPR7 ligand A.

[1025] [SEQ ID NO:2]

[1026] This shows the amino acid sequence of mouse GPR7 ligand A.

[1027] [SEQ ID NO:3]

[1028] This shows the amino acid sequence of rat GPR7 ligand A.

[1029] [SEQ ID NO:4]

[1030] This shows the amino acid sequence of human GPR7 ligand B.

[1031] [SEQ ID NO:5]

[1032] This shows the amino acid sequence of mouse GPR7 ligand B.

[1033] [SEQ ID NO:6]

[1034] This shows the amino acid sequence of rat GPR7 ligand B.

[1035] [SEQ ID NO:7]

[1036] This shows the amino acid sequence of human GPR7 ligand C.

[1037] [SEQ ID NO:8]

[1038] This shows the amino acid sequence of human GPR7 ligand D.

[1039] [SEQ ID NO:9]

[1040] This shows the amino acid sequence of mouse GPR7 ligand C.

[1041] [SEQ ID NO:10]

[1042] This shows the amino acid sequence of mouse GPR7 ligand D.

[1043] [SEQ ID NO:11]

[1044] This shows the amino acid sequence of rat GPR7 ligand C.

[1045] [SEQ ID NO:12]

[1046] This shows the amino acid sequence of rat GPR7 ligand D.

[1047] [SEQ ID NO:13]

[1048] This shows the amino acid sequence of human GPR7 ligand E.

[1049] [SEQ ID NO:14]

[1050] This shows the amino acid sequence of mouse GPR7 ligand E.

[1051] [SEQ ID NO:15]

[1052] This shows the amino acid sequence of rat GPR7 ligand E.

[1053] [SEQ ID NO:16]

[1054] This shows the amino acid sequence of human GPR7 ligand F.

[1055] [SEQ ID NO:17]

[1056] This shows the amino acid sequence of mouse GPR7 ligand F.

[1057] [SEQ ID NO:18]

[1058] This shows the amino acid sequence of rat GPR7 ligand F.

[1059] [SEQ ID NO:19]

[1060] This shows the amino acid sequence of human GPR7 ligand precursorG containing no secretory signal.

[1061] [SEQ ID NO:20]

[1062] This shows the amino acid sequence of mouse GPR7 ligand precursorG containing no secretory signal.

[1063] [SEQ ID NO:21]

[1064] This shows the amino acid sequence of rat GPR7 ligand precursor Gcontaining no secretory signal.

[1065] [SEQ ID NO:22]

[1066] This shows the amino acid sequence of human GPR7 ligand precursorH containing a secretory signal.

[1067] [SEQ ID NO:23]

[1068] This shows the amino acid sequence of mouse GPR7 ligand precursorH containing a secretory signal.

[1069] [SEQ ID NO:24]

[1070] This shows the amino acid sequence of rat GPR7 ligand precursor Hcontaining a secretory signal.

[1071] [SEQ ID NO:25]

[1072] This shows the base sequence of DNA encoding human GPR7 ligand A.

[1073] [SEQ ID NO:26]

[1074] This shows the base sequence of DNA encoding mouse GPR7 ligand A.

[1075] [SEQ ID NO:27]

[1076] This shows the base sequence of DNA encoding rat GPR7 ligand A.

[1077] [SEQ ID NO:28]

[1078] This shows the base sequence of DNA encoding human GPR7 ligand B.

[1079] [SEQ ID NO:29]

[1080] This shows the base sequence of DNA encoding mouse GPR7 ligand B.

[1081] [SEQ ID NO:30]

[1082] This shows the base sequence of DNA encoding rat GPR7 ligand B.

[1083] [SEQ ID NO:31]

[1084] This shows the base sequence of DNA encoding human GPR7 ligand C.

[1085] [SEQ ID NO:32]

[1086] This shows the base sequence of DNA encoding human GPR7 ligand D.

[1087] [SEQ ID NO:33]

[1088] This shows the base sequence of DNA encoding mouse GPR7 ligand C.

[1089] [SEQ ID NO:34]

[1090] This shows the base sequence of DNA encoding mouse GPR7 ligand D.

[1091] [SEQ ID NO:35]

[1092] This shows the base sequence of DNA encoding rat GPR7 ligand C.

[1093] [SEQ ID NO:36]

[1094] This shows the base sequence of DNA encoding rat GPR7 ligand D.

[1095] [SEQ ID NO:37]

[1096] This shows the base sequence of DNA encoding human GPR7 ligand E.

[1097] [SEQ ID NO:38]

[1098] This shows the base sequence of DNA encoding mouse GPR7 ligand E.

[1099] [SEQ ID NO:39]

[1100] This shows the base sequence of DNA encoding rat GPR7 ligand E.

[1101] [SEQ ID NO:40]

[1102] This shows the base sequence of DNA encoding human GPR7 ligand F.

[1103] [SEQ ID NO:41]

[1104] This shows the base sequence of DNA encoding mouse GPR7 ligand F.

[1105] [SEQ ID NO:42]

[1106] This shows the base sequence of DNA encoding rat GPR7 ligand F.

[1107] [SEQ ID NO:43]

[1108] This shows the base sequence of DNA encoding human GPR7 ligandprecursor G containing no secretory signal.

[1109] [SEQ ID NO:44]

[1110] This shows the base sequence of DNA encoding mouse GPR7 ligandprecursor G containing no secretory signal.

[1111] [SEQ ID NO:45]

[1112] This shows the base sequence of DNA encoding rat GPR1 ligandprecursor G containing no secretory signal.

[1113] [SEQ ID NO:46]

[1114] This shows the base sequence of DNA encoding human GPR7 ligandprecursor H containing a secretory signal.

[1115] [SEQ ID NO:47]

[1116] This shows the base sequence of DNA encoding mouse GPR7 ligandprecursor H containing a secretory signal.

[1117] [SEQ ID NO:48]

[1118] This shows the base sequence of DNA encoding rat GPR7 ligandprecursor H containing a secretory signal.

[1119] [SEQ ID NO:49]

[1120] This shows the amino acid sequence of human GPR7.

[1121] [SEQ ID NO:50]

[1122] This shows the base sequence of a DNA containing the DNA encodinghuman GPR7.

[1123] [SEQ ID NO:51]

[1124] This shows a synthetic DNA used in EXAMPLE 1 to screen cDNAencoding human GPR7 ligand precursor H.

[1125] [SEQ ID NO:52]

[1126] This shows a synthetic DNA used in EXAMPLE 1 to screen cDNAencoding human GPR7 ligand precursor H.

[1127] [SEQ ID NO:53]

[1128] This shows a synthetic DNA used in EXAMPLE 2 to screen cDNAencoding mouse GPR7 ligand precursor H.

[1129] [SEQ ID NO:54]

[1130] This shows a synthetic DNA used in EXAMPLE 2 to screen cDNAencoding mouse GPR7 ligand precursor H.

[1131] [SEQ ID NO:55]

[1132] This shows a synthetic DNA used in EXAMPLE. 3 to screen cDNAencoding rat GPR7 ligand precursor H.

[1133] [SEQ ID NO:56]

[1134] This shows a synthetic DNA used in EXAMPLE 3 to screen cDNAencoding rat GPR7 ligand precursor H.

[1135] [SEQ ID NO:57]

[1136] This shows the base sequence of a primer used in REFERENCEEXAMPLE 1.

[1137] [SEQ ID NO:58]

[1138] This shows the base sequence of a primer used in REFERENCEEXAMPLE 1.

[1139] [SEQ ID NO:59]

[1140] This shows the amino acid sequence of rat TGR26.

[1141] [SEQ ID NO:60]

[1142] This shows the base sequence of DNA encoding rat TGR26.

[1143] [SEQ ID NO:61]

[1144] This shows the base sequence of Primer 1 used for PCR inREFERENCE EXAMPLE 3.

[1145] [SEQ ID NO:62]

[1146] This shows the base sequence of Primer 2 used for PCR inREFERENCE EXAMPLE 3.

[1147] [SEQ ID NO:63]

[1148] This shows the base sequence of a primer used in EXAMPLE 9.

[1149] [SEQ ID NO:64]

[1150] This shows the base sequence of a primer used in EXAMPLE 9.

[1151] [SEQ ID NO:65]

[1152] This shows the base sequence of a primer used in EXAMPLE 9.

[1153] [SEQ ID NO:66]

[1154] This shows the amino acid sequence of bovine GPR7 ligand A.

[1155] [SEQ ID NO:67]

[1156] This shows the amino acid sequence of bovine GPR7 ligand B.

[1157] [SEQ ID NO:68]

[1158] This shows the amino acid sequence of bovine GPR7 ligand C.

[1159] [SEQ ID NO:69]

[1160] This shows the amino acid sequence of bovine GPR7 ligand D.

[1161] [SEQ ID NO:70]

[1162] This shows the amino acid sequence of bovine GPR7 ligand E.

[1163] [SEQ ID NO:71]

[1164] This shows the amino acid sequence of bovine GPR7 ligand F.

[1165] [SEQ ID NO:72]

[1166] This shows the amino acid sequence of bovine GPR7 ligandprecursor G containing no secretory signal.

[1167] [SEQ ID NO:73]

[1168] This shows the amino acid sequence of bovine GPR7 ligandprecursor H containing a secretory signal.

[1169] [SEQ ID NO:74]

[1170] This shows the base sequence of DNA encoding bovine GPR7 ligandA.

[1171] [SEQ ID NO:75]

[1172] This shows the base sequence of DNA encoding bovine GPR7 ligandB.

[1173] [SEQ ID NO:76]

[1174] This shows the base sequence of DNA encoding bovine GPR7 ligandC.

[1175] [SEQ ID NO:77]

[1176] This shows the base sequence of DNA encoding bovine GPR7 ligandD.

[1177] [SEQ ID NO:78]

[1178] This shows the base sequence of DNA encoding bovine GPR7 ligandE.

[1179] [SEQ ID NO:79]

[1180] This shows the base sequence of DNA encoding bovine GPR7 ligandF.

[1181] [SEQ ID NO:80]

[1182] This shows the base sequence of DNA encoding bovine GPR7 ligandprecursor G containing no secretory signal.

[1183] [SEQ ID NO:81]

[1184] This shows the base sequence of DNA encoding bovine GPR7 ligandprecursor H containing a secretory signal.

[1185] [SEQ ID NO:82]

[1186] This shows the base sequence of a primer used in EXAMPLE 12.

[1187] [SEQ ID NO:83]

[1188] This shows the base sequence of a primer used in EXAMPLE 12.

[1189] [SEQ ID NO:84]

[1190] This shows the amino acid sequence of human GPR8.

[1191] [SEQ ID NO:85]

[1192] This shows the base sequence of a DNA containing the DNA encodinghuman GPR8.

[1193] [SEQ ID NO:86]

[1194] This shows the amino acid sequence of bovine GPR7.

[1195] [SEQ ID NO:87]

[1196] This shows the base sequence of a DNA containing the DNA encodingbovine GPR7.

[1197] [SEQ ID NO:88]

[1198] This shows the amino acid sequence of bovine GPR8.

[1199] [SEQ ID NO:89]

[1200] This shows the base sequence of a DNA containing the DNA encodingbovine GPR8.

[1201] [SEQ ID NO:90]

[1202] This shows the base sequence of a primer used in EXAMPLE 16.

[1203] [SEQ ID NO:91]

[1204] This shows the base sequence of a primer used in EXAMPLE 16.

[1205] [SEQ ID NO:92]

[1206] This shows the base sequence of a primer used in EXAMPLE 16.

[1207] [SEQ ID NO:93]

[1208] This shows the base sequence of a primer used in EXAMPLE 17.

[1209] [SEQ ID NO:94]

[1210] This shows the base sequence of a primer used in EXAMPLE 17.

[1211] [SEQ ID NO:95]

[1212] This shows the base sequence of a primer used in EXAMPLE 17.

[1213] [SEQ ID NO:96]

[1214] This shows the base sequence of a primer used in EXAMPLE 18.

[1215] [SEQ ID NO:97]

[1216] This shows the base sequence of a primer used in EXAMPLE 18.

[1217] [SEQ ID NO:98]

[1218] This shows the base sequence of a primer used in EXAMPLE 19.

[1219] [SEQ ID NO:99]

[1220] This shows the base sequence of a primer used in EXAMPLE 19.

[1221] [SEQ ID NO:100]

[1222] This shows the amino acid sequence of human GPR8 ligand (1-23).

[1223] Transformant Escherichia coli JM109/pTAhGPR7-1, which wasobtained in EXAMPLE 1 later described, has been deposited asJM109/pTAhGPR7L-1 since Jun. 27, 2001 on the National Institute ofAdvanced Industrial Science and Technology, International PatentOrganism Depositary, located at Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan (postal code 305-8566), under the Accession Number FERMBP-7640, and since on Jun. 19, 2001 on the Institute for Fermentation(IFO), located at 2-17-85, Juso Honcho, Yodogawa-ku, Osaka-shi, Osaka,Japan (postal code 532-8686), under the Accession Number IFO 16644,respectively.

[1224] Transformant Escherichia coli JM109/pTAmGPR7-1, which wasobtained in EXAMPLE 2 later described, has been deposited asJM109/pTAmGPR7L-1 since Jun. 27, 2001 on the National Institute ofAdvanced Industrial Science and Technology, International PatentOrganism Depositary under the Accession Number FERM BP-7641, and sinceon Jun. 19, 2001 on the Institute for Fermentation (IFO) under theAccession Number IFO 16656, respectively.

[1225] Transformant Escherichia coli JM109/pTArGPR7-1, which wasobtained in EXAMPLE 3 later described, has been deposited asJM109/pTArGPR7L-1 since Jun. 27, 2001 on the National Institute ofAdvanced Industrial Science and Technology, International PatentOrganism Depositary under the Accession Number FERM BP-7642, and sinceon Jun. 19, 2001 on the Institute for Fermentation (IFO) under theAccession Number IFO 16657, respectively.

[1226] Transformant Escherichia coli JM109/pTAbGPR7L-1, which wasobtained in EXAMPLE 12 later described, has been deposited since Dec.17, 2001 on the National Institute of Advanced. Industrial Science andTechnology, International Patent Organism Depositary under the AccessionNumber FERM BP-7829, and since on Dec. 6, 2001 on the Institute forFermentation (IFO) under the Accession Number IFO. 16736, respectively.

[1227] Transformant Escherichia coli JM109/pTAbGPR7, which was obtainedin EXAMPLE 18 later described, has been deposited since May 24, 2002 onthe National Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary under the Accession Number FERMBP-8050.

[1228] Transformant Escherichia coli JM109/pTAbGPR8, which was obtainedin EXAMPLE 19 later described, has been deposited since May 24, 2002 onthe National Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary under the Accession Number FERMBP-8051.

[1229] Transformant Escherichia coli DH10B/pAK-rGPR7, which was obtainedin REFERENCE EXAMPLE 3 later described, has been deposited since Oct.31, 2000 on the Institute for Fermentation (IFO), located at 2-17-85,Juso Honcho, Yodogawa-ku, Osaka-shi, Osaka, Japan, under the AccessionNumber IFO 16496 and since on Nov. 13, 2000 on the Ministry ofInternational Trade and Industry, Agency of Industrial Science andTechnology, National Institute of Bioscience and Human Technology(NIBH), located at 1-1-3 Higashi, Tsukuba, Ibaraki, Japan, under theAccession Number FERM BP-7365, respectively.

EXAMPLES

[1230] The present invention will be described in more detail below,with reference to REFERENCE EXAMPLES and EXAMPLES, but is not deemed tolimit the scope of the present invention thereto.

Reference Example 1

[1231] Amplification of Human GPR7 DNA by PCR using Human ChromosomalDNA

[1232] By use of human chromosomal DNA as a template, amplification ofDNA was carried out by PCR using two synthetic primers (SEQ ID NO:57 andSEQ ID NO:58). The synthetic primers were constructed so as to amplifythe gene in the region to be translated to its receptor protein wasamplified. In this case, the recognition sequences for restrictionenzymes were added at the 5′ and 3′ ends, respectively, so that the basesequences recognized by restriction enzymes ClaI and SpeI were added tothe gene at the 5′ and 3′ ends, respectively. The reaction solution wascomposed of 0.5 μl of human chromosomal DNA (TaKaRa Shuzo Co., Ltd.), 1μM each of the synthetic DNA primers, 0.8 mM dNTPs, 1 mM MgCl₂ and 1 μlof KOD polymerase (Toyobo Co., Ltd.), to which the buffer attached tothe enzymes was added to make the total reaction volume of 50 μl. Foramplification, after heating at 94° C. for 60 seconds, the cycle set toinclude 98° C. for 15 seconds, 65° C. for 2 seconds and 72° C. for 30seconds was repeated 35 times, using Thermal Cycler (TaKaRa Shuzo Co.,Ltd.). The amplified product was confirmed by 0.8% agarose gelelectrophoresis followed by staining with ethidium bromide.

Reference Example 2

[1233] Subcloning of the PCR Product into a Plasmid Vector andConfirmation of the Amplified DNA Sequence by Decoding the Base Sequenceof the Inserted DNA Part

[1234] The reaction solution of PCR performed in REFERENCE EXAMPLE 1 wasseparated by 0.8% low melting point agarose gel electrophoresis. Theband part was excised with a razor blade, ground into small pieces,extracted with phenol and then with phenol/chloroform and precipitatedin ethanol to recover DNAs. According to the protocol attached toPCR-Script™ Amp SK(+) Cloning Kit (Stratagene Co.), the recovered DNAswere subcloned to plasmid vector pCR-Script Amp SK(+). The recombinantvector was introduced into Escherichia coli DH5α competent cells (ToyoboCo., Ltd.) to produce transformants. The clones bearing the DNA-insertedfragment were selected in an LB agar medium supplemented withampicillin, IPTG and X-gal. Only the clones exhibiting white color werepicked up with a sterilized toothpick to obtain transformant E. coliDH5α/GPR7. The individual clones were cultured overnight in an LB mediumcontaining ampicillin. Plasmid DNAs were prepared using QIAwell 8Plasmid Kit (QIAGEN, Inc.). An aliquot of the DNAs thus prepared wascleaved by restriction enzymes ClaI and SpeI to confirm the size of thereceptor cDNA fragment inserted. The reaction for base sequencing wascarried out by using a DyeDeoxy Terminator Cycle Sequence Kit (AppliedBiosystems, Inc.), followed by decoding with a fluorescent automaticsequencer (SEQ ID NO:50). The pCR-Script Amp SK(+) plasmid bearing theDNA having the base sequence represented by SEQ ID NO:50 was namedpCR-Script human GPR7. The amino acid sequence of human GPR7 encoded bythe DNA having the base sequence represented by SEQ ID NO:50 ispresented in SEQ ID NO:49. The DNA sequence of human GPR7 sequencedherein differed by 2 bases from the DNA sequence reported by O'Dowd etal. (O'Dowd, B. F. et al., Genomics, 28, 84-91, 1995). These basescorresponding to 893rd and 894th bases in SEQ ID NO:50 were reportedly Cand G in the report by O'Dowd et al., whereas they were found to be Gand C in this REFERENCE EXAMPLE. Based on the findings, the 296th aminoacid of SEQ ID NO:49 in the translated amino acid sequence is Ser inthis EXAMPLE, which was reportedly Thr in O'Dowd et al.

Reference Example 3

[1235] Cloning of cDNA Encoding Rat Whole Brain-Derived GProtein-Coupled Receptor Protein and Base Sequencing

[1236] By use of rat whole brain cDNA (CLONTECH Labs. Inc.), PCR wascarried out using two primers, Primer 1 (SEQ ID NO:61) and Primer 2 (SEQID NO:62) designed from the base sequence of DNA encoding human GPR8.The reaction solution for PCR wherein {fraction (1/10)} volume of theabove cDNA was used as a template, was composed of {fraction (1/50)}volume of Advantage-2 cDNA Polymerase Mix (CLONTECH Labs. Inc.), 0.2 μMof Primer 3, 0.2 μM of Primer 2 and 200 μM of dNTPs, to which the bufferattached to the enzymes was added to make the total volume of 25 μl. PCRwas carried out, (1) after heating at 94° C. for 2 minutes, by repeating(2) the cycle set to include 94° C. for 20 seconds and 72° C. for 2minutes 3 times, (3) the cycle set to include 94° C. for 20 seconds, 66°C. for 20 seconds and 68° C. for 2 minutes 3 times and (4) the cycle setto include 94° C. for 20 seconds, 60° C. for 20 seconds and 68° C. for 2minutes 36 times, finally followed by extension at 68° C. for 7 minutes.The reaction product after the PCR was subcloned to pCR2.1-TOPO(Invitrogen, Inc.) in accordance with the protocol of TA Cloning Kitvector using TA cloning kit (Invitrogen, Inc.). After introducing intoEscherichia coli DH5α, clones bearing cDNA were selected in LB agarmedium supplemented with ampicillin. Individual clones were sequenced toacquire the base sequence (SEQ ID NO:60) of cDNA encoding novel Gprotein-coupled receptor protein. The novel G protein-coupled receptorprotein containing the amino acid sequence (SEQ ID NO:59) encoded by thebase sequence of this DNA was named TGR26.

[1237] The amino acid sequence represented by SEQ ID NO:59 had 84.8%homology to GPR7 (Genomics, 28, 84-91, 1995), which is known human Gprotein-coupled receptor protein.

[1238] From the above transformants bearing the plasmid inserted withthe TGR26-encoding DNA, one clone was selected and shake cultured in LBmedium supplemented with ampicillin to acquire plasmid. The plasmid wastreated with restriction enzymes ClaI and SpeI to excise the insertencoding TGR26. Using pAKKO-1.11H, which was similarly treated withrestriction enzymes ClaI and SpeI, and Ligation Express Kit (CLONTECH,Inc.), ligation was performed and the ligated product was transfected toEscherichia coli DH10B by electroporation. With respect to the clonethus obtained, the structure of the plasmid for expression cellconstruction was confirmed by the treatment with restriction enzymes andsequencing. The clone was then named Escherichia coli DH10B/pAK-rGPR7.

Reference Example 4

[1239] Preparation of TGR26-Expressing CHO Cells

[1240] After Escherichia coli DH5α (Toyobo Co., Ltd.) transformed by theexpression plasmid pAK-rGPR7 described in REFERENCE EXAMPLE 3 wascultured, pAK-rGPR7 plasmid DNA was prepared. The plasmid DNA wastransfected to CHO dhfr⁻ cells by using CellPhect Transfection Kit(Amersham Pharmacia Biotech), according to the protocol attached. Aco-precipitated suspension of 5 μg of DNA and calcium phosphate wasadded to 2 Petri dishes of 6 cm diameter, on which 3×10⁵ CHO dhfr⁻ cellshad been plated 48 hours before. After cultivation for a day in MEM amedium containing 10% fetal cow serum, the cells were passaged andcultured in nucleic acid-free MEM a medium containing 10% dialysis fetalcow serum as a selection medium. From the colony of the transformantwhich was TGR26-expressing CHO cells grown in the selection medium, 44clones were selected.

Example 1

[1241] Acquisition of GPR7 Ligand Precursor Gene from Human Whole BraincDNA by PCR and Construction of Expression Plasmid

[1242] Using human whole brain cDNA purchased from CLONTECH as atemplate, amplification was performed by PCR using the following twosynthetic DNAs. GSF1: 5′-GTCGACATGGCCCGGTCCGCGACACT (SEQ ID NO:51)GGCGGCC-3′ GSR2: 5′-GCTAGCAGCGGTGCCAGGAGAGGTCC (SEQ ID NO:52) GGGCTCA-3′

[1243] The reaction solution for PCR contained 1 μl of cDNA solution,0.5 μl of GSF1 (10 μM), 0.5 μl of GSR2 (10 μM), 2.5 μl of 10× reactionsolution attached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq(CLONTECH, Inc.), to which 17.5 μl of Otsuka distilled water was addedto make 25 μl in total. The reaction solution was applied to PCR usingThermal Cycler 9600. The conditions for PCR were set forth: afterdenaturation at 95° C. for 2 minutes, the cycle set to include 98° C.for 10 seconds, 60° C. for 20 seconds and 72° C. for 20 seconds wasrepeated 35 times. After it was confirmed by electrophoresis using analiquot of the PCR product that the PCR product of about 400 bp wasamplified, the PCR product was purified using Quiagen PCR purificationKit and directly sequenced to obtain the sequence shown by FIG. 1. Theamino acid sequence deduced from the DNA sequence of FIG. 1 was thesequence shown in FIG. 2. Next, the PCR product recovered from the gelwas subcloned to Escherichia coli JM109 using TA Cloning Kit(Invitrogen, Inc.), to acquire Escherichia coli JM109/pTAhGPR7-1.Plasmid pTAhGPR7-1 was extracted from Escherichia coli obtained by thesubcloning, using a plasmid extractor (Kurabo Co., Ltd.) to identify thebase sequence of the inserted fragment. It was confirmed that thesequence was the same as that of human GPR7 ligand cDNA shown in FIG. 1.Next, after digestion with restriction enzymes SalI and NheI, human GPR7ligand cDNA fragment of about 0.4 kb was obtained from the plasmid.Furthermore, expression vector pAKKO-111H for animal cells was digestedby restriction enzymes sites SalI and NheI of the multi-cloning sites,and electrophoresed to recover the vector portion. The human GPR7 ligandcDNA fragment prepared by the foregoing procedures was ligated throughligation and Escherichia coli JM109 was transformed to obtainEscherichia coli JM109 to acquire E. coli JM109/pAK-S64.

[1244] Transformant E. coli JM109/pAK-S64 was cultured to prepare theDNA of plasmid pAK-S64 in large quantities.

Example 2

[1245] Acquisition of GPR7 Ligand Precursor Gene from Mouse Whole BraincDNA by PCR

[1246] Using mouse whole brain cDNA as a template, amplification wasperformed by PCR using the following two synthetic DNAs. MFSAL1:5′-GTCGACAGCTCCATGGCCCGGTGTAGGACGCTG-3′ (SEQ ID NO:53) MRNHE1:5′-GCTAGCTCAGGTGCTCTGGCAATCAGTCTCGTG-3′ (SEQ ID NO:54)

[1247] The reaction solution for PCR contained 1 μl of cDNA solution,0.5 μl of MFSAL1 (10 μM), 0.5 μl of MRNHE1 (10 μM), 2.5 μl of 10×reaction solution attached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq(CLONTECH, Inc.), to which 17.5 μl of Otsuka distilled water was addedto make 25 μl in total. The reaction solution was applied to PCR usingThermal Cycler 9600. The conditions for PCR were set forth: afterdenaturation at 95° C. for 2 minutes, the cycle set to include 98° C.for 10 seconds, 60° C. for 20 seconds and 72° C. for 20 seconds wasrepeated 35 times. After it was confirmed by electrophoresis using analiquot of the PCR product that the PCR product of about 400 bp wasamplified, the PCR product was purified using Quiagen PCR purificationKit and directly sequenced to obtain the sequence shown by FIG. 3. Theamino acid sequence deduced from the DNA sequence of FIG. 3 was thesequence shown in FIG. 4. Next, the PCR product recovered from the gelwas subcloned to Escherichia coli JM109 using TA Cloning Kit(Invitrogen, Inc.) to acquire Escherichia coli JM109/pTAmGPR7-1. PlasmidpTAmGPR7-1 was extracted from Escherichia coli obtained by thesubcloning, using a plasmid extractor (Kurabo Co., Ltd.) to identify thebase sequence of the inserted fragment. It was confirmed that thesequence was the same as that of mouse GPR7 ligand cDNA shown in FIG. 3.

Example 3

[1248] Acquisition of GPR7 Ligand Precursor Gene from Rat Whole BraincDNA by PCR

[1249] Using rat whole brain cDNA as a template, amplification wasperformed by PCR using the following two synthetic DNAs. RF:5′-CACGGCTCCATGGTCCGGTGTAGGACG-3′ (SEQ ID NO:55) RR:5′-CAGCGTCGAGGTTTGGGTTGGGGTTCA-3′ (SEQ ID NO:56)

[1250] The reaction solution for PCR contained 1 μl of cDNA solution,0.5 μl of RF (10 μM), 0.5 μl of RR (10 μM), 2.5 μl of 10× reactionsolution attached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq(CLONTECH, Inc.), to which 17.5 μl of Otsuka distilled water was addedto make 25 μl in total. The reaction solution was applied to PCR usingThermal Cycler 9600. The conditions for PCR were set forth: afterdenaturation at 95° C. for 2 minutes, the cycle set to include 98° C.for 10 seconds, 60° C. for 20 seconds and 72° C. for 20 seconds wasrepeated 35 times. After it was confirmed by electrophoresis using analiquot of the PCR product that the PCR product of about 400 bp wasamplified, the PCR product was purified using Quiagen PCR purificationKit and directly sequenced to obtain the sequence shown by FIG. 5. Theamino acid sequence deduced from the DNA sequence of FIG. 5 was thesequence shown in FIG. 6. Next, the PCR product recovered from the gelwas subcloned to Escherichia coli JM109 using TA Cloning Kit(Invitrogen, Inc.) to acquire Escherichia coli JM109/pTArGPR7-1. PlasmidpTArGPR7-1 was extracted from Escherichia coli obtained by thesubcloning, using a plasmid extractor (Kurabo Co., Ltd.) to identify thebase sequence of the inserted fragment. It was confirmed that thesequence was the same as that of mouse GPR7 ligand cDNA shown in FIG. 5.

Example 4

[1251] Transient Expression of GPR7 Expression Plasmid and ReporterPlasmid in Chinese Hamster Ovary (CHO) Cells

[1252]Escherichia coli JM109 was transformed using a plasmid prepared byinserting human GPR7 DNA obtained in REFERENCE EXAMPLE 2 into expressionplasmid pAKKO-111H for animal cells by a publicly known method. Afterthe colony obtained was isolated and cultured, GPR7 expression plasmidDNA was prepared using QUIAGEN Plasmid Maxi Kit (QIAGEN, Inc.). Also,plasmid DNA of pCRE-Luc (CLONTECH, Inc.), in which luciferase gene wasligated as a reporter at the downstream of cAMP response element (CRE),was prepared in a similar manner.

[1253] GPR7 expression plasmid and pCRE-Luc were transiently expressedin CHO cells, to which the expression vector inserted with no receptorgene was transfected. The CHO cells were plated on a 96-well plate(Corning Costar, Inc.) in 40,000 cells/well in a medium volume of 100μl, followed by incubation overnight at 37° C. For incubation on theplate, DMEM (Dulbecco's modified Eagle's medium, Gibco BRL, Inc.)supplemented with 10% fetal cow serum only was used as medium.

[1254] Each plasmid was diluted to a concentration of 240 ng/μl andadded to 240 μl of Opti-MEM-I (Gibco BRL, Inc.) in a ratio of 9 μl ofGPR7 expression plasmid to 1 μl of pCRE-Luc. The mixture was mixed withan equal volume of a mixture obtained by adding 10 μl of Lipofectamine2000 (Gibco BRL, Inc.) to 240 μl of Opti-MEM-I (Gibco BRL, Inc.) in asimilar manner to produce the liposome-plasmid complex in accordancewith the instruction manual attached to Lipofectamine 2000. The complexwas added in 25 μl each/well to the culture medium of CHO cells. Fourhours later, the culture medium was replaced by an assay buffer (DMEMsupplemented with 0.1% bovine serum albumin) to make the mediumserum-free, followed by incubation overnight at 37° C.

Example 5

[1255] Expression of Ligand Gene in CHO Cells

[1256] The human ligand cDNA-inserted expression plasmid pAK-S64 foranimal cells prepared in EXAMPLE 1 was transiently expressed in CHOcells in a manner similar to EXAMPLE 4, except that the cells wereplated on a 6-well plate (Falcon Corp.) in 600,000 cells/well andincubated overnight, and then the ligand gene plasmid was introduced.The plasmid was diluted to a concentration of 240 ng/μl and an aliquotof 10 μl was added to 240 μl of Opti-MEM-I. The mixture was mixed withan equal volume of a mixture obtained in a similar manner by adding 10μl of Lipofectamine 2000 to 240 μl of Opti-MEM-I to produce theliposome-plasmid complex in accordance with the method described in theinstruction manual attached to Lipofectamine 2000. Each of them wasadded in 500 μl each/well to the culture medium of CHO cells. Four hourslater, the culture medium was replaced by the assay buffer to make themedium serum-free. The medium of each well was recovered 18 hours afterthe medium exchange to obtain the CHO cell culture supernatantcontaining the ligand peptide.

Example 6

[1257] Detection of Luciferase Activity Suppression by S64 ExpressionCell Supernatant in CHO Cells with Transient Expression of GPR7

[1258] The pAK-S64 expression culture supernatant prepared in EXAMPLE 5and forskolin in the final concentration of 2 μM were added to theculture medium of CHO cells wherein GPR7 was transiently expressedaccording to the procedures of EXAMPLE 4. The culture supernatant of CHOcells wherein a ligand gene-free empty expression vector (pAKKO-111H)was transiently expressed according to the procedures of EXAMPLE 5 wasadded as well. At this stage, the expression supernatant was diluted to2-, 4-, 8- and 16-fold with the assay buffer. After the addition of thesupernatant, incubation was carried out for 4 hours at 37° C. to causethe promotion or suppression of transcription/translation of thereporter (luciferase) gene via intracellular signal transduction inducedby the agonist activity of ligand mediated by the receptor. Aftercompletion of the incubation, the assay buffer was removed from eachwell and 50 μl each of PicaGene LT2.0 (Toyo Ink Mfg. Co., Ltd.) as aluminescent substrate was added to the well. After the cells were lysedand thoroughly mixed with the substrate, the luminescence amountassociated with the expression induction level of the reporter gene ineach well was assayed by using a plate reader (ARVOsx Multi-labelCounter, Perkin Elmer, Inc.). As the result, the expression suppressionof the reporter gene was detected as a decreased luciferase activityonly when the culture supernatant of pAK-S64 was added (FIG. 8).Moreover, the degree of this suppression was dependent on theconcentration of the pAK-S64 culture-supernatant. This indicates thatthe product expressed by the plasmid inserted into pAK-S64 transducedthe intracellular signal mediated by GPR7, i.e., acted as a ligand toGPR7.

Example 7

[1259] Detection of Luciferase Activity Suppression by S64 ExpressionCell Supernatant in CHO Cells with Transient Expression of TGR26

[1260] The TGR26 expression plasmid DNA was prepared in a manner similarto EXAMPLE 4, using expression plasmid pAKKO-111H for animal cells inwhich TGR26 DNA obtained in REFERENCE EXAMPLE 3 was inserted by publiclyknown methods. The plasmid DNA and the luciferase gene were likewiseexpressed transiently in the CHO cells according to the procedures ofEXAMPLE 4. To the cells, the pAK-S64 expression culture supernatantprepared in EXAMPLE 5 and the culture supernatant of cells wherein theempty expression vector alone was expressed, were added and afterforskolin was further added thereto in the final concentration of 2 μM,it was attempted to detect the ligand activity in a manner similar toEXAMPLE 6. As the result, the pAK-S64 supernatant decreasedconcentration-dependently the luciferase activity increased by forskolin(FIG. 9).

Example 8

[1261] Suppression of cAMP Production Level in GPR7-Expressed CHO Cellsby S64 Expression Cell Supernatant

[1262] Using the plasmid for GPR7 expression prepared in EXAMPLE 4,CHO-GPR7 as CHO cells capable of stably expressing GPR7 was prepared bypublicly known methods. Mock CHO cells were plated on a 96-well plate(Beckton-Dickinson, Inc.) in 20,000 cells/well. After incubationovernight at 37° C. under 5% CO₂, the culture medium was used for theassay. The sample buffer used was Dulbecco's modified Eagle's medium(DMEM, Gibco BRL, Inc.) supplemented with 0.1% bovine serum albumin(Sigma) and 0.2 mM IBMX (Sigma). The cells were washed twice with thesample buffer. After pre-incubation at 37° C. for 30 minutes under 5%CO₂, the cells were further washed twice and the sample was addedthereto followed by incubation at 37° C. for 30 minutes under 5% CO₂.The cells were further washed twice and the sample was added thereto at37° C. for 30 minutes. Four kinds of the samples were the sample bufferalone (intact), 1 μM of forskolin (Wako Pure Chemical Industries, Ltd.)as a reagent to stimulate an increase of cAMP production, simultaneousaddition of the CHO cell culture supernatant obtained in EXAMPLE 5prepared by transient expression of pAK-S64 (S64 supernatant) andforskolin, and simultaneous addition of the culture supernatant ofpAKKO-111H-expressed CHO cells (pAKKO supernatant) and forskolin. Afterincubation in the presence of the sample, the intracellular cAMPproduction level was assayed by using cAMP Screen System (ABI). Theresults revealed that the intracellular cAMP production level wasCHO-GPR7-specifically suppressed by adding the S64 supernatant (FIG. 10)and no suppression of the intracellular cAMP production level was notedin the mock CHO cells (FIG. 11).

Example 9

[1263] Study of Tissue Distribution of GPR7 Ligand mRNA in Rat by RT-PCR

[1264] Various organs were withdrawn from Wistar rat. The total RNA andpoly(A)⁺ RNA were prepared using Isogen (Nippon Gene Co., Ltd.) and mRNApurification kit (Pharmacia), respectively, according to the respectiveinstruction manuals. After 1 μg of poly(A)⁺ RNA was digested with DnaseI(Amplification Grade, Gibco BRL, Inc.), a 160 ng aliquot was treated at42° C. using RNA PCR Kit (TaKaRa Shuzo Co., Ltd.) according to theinstruction manual to synthesize cDNA. The cDNA synthesized was made asolution of 4 ng/μl when calculated as poly(A)⁺ RNA and used as atemplate for RT-PCR thereafter. Using Sequence Detection System Prism7700 (PE Biosystems), RT-PCR was carried out, wherein primers:5′-CTGTCGAGTTTCCACAGGTTCC-3′ (SEQ ID NO:63) and5′-TTGCGCAGAGGTACGGTTCC-3′ (SEQ ID NO:64) were used for amplificationand detection, and 5′-(Fam)-CGTGCCAAGAAACGCGTGACCTTGTT-(Tamra)-3′ (SEQID NO:65) was used as TaqMan probe. In the reaction solution for RT-PCR,0.05 μl each of 100 μM primer solution, 0.5 μl of 5 μM TaqMan probe, 2.5μl of 10× reaction solution attached, 2.5 μl of dNTP (10 mM) and 0.5 μlof the cDNA solution prepared above were added to 12.5 μl of TaqManUniversal PCR Master Mix (PE Biosystems), to which distilled water wasadded to make the total solution volume 25 μl. The reaction solution wasapplied to PCR using Thermal Cycler 9600. After denaturation at 50° C.for 2 minutes and 95° C. for 10 minutes, PCR was carried out byrepeating 40 times the cycle set to include 95° C. for 15 seconds and60° C. for 1 minute. The expression level of GPR7 ligand mRNA in thevarious tissues in rat was assessed in terms of the copy number per 1 ngof poly(A)⁺ RNA (FIG. 12).

Example 10

[1265] Purification of Endogenous GPR7 Ligand from Bovine Hypothalamus.

[1266] Since it was found that human GPR7 ligand precursor mRNA wasabundantly expressed in hypothalamus and spinal cord, the endogenousGPR7 ligand was purified from bovine hypothalamus as the startingmaterial, using human GPR7-expressed CHO cells. The purification wasperformed using as an indicator the intracellular cAMP productionsuppressing activity (as determined using cAMP-Screen System (ABI)).

[1267] First, 1.0 kg of bovine hypothalamus in a frozen state was boiledin Milli-Q Water. After cooling, acetic acid was added to become 1M,which was then homogenized with a polytron. After agitation overnight,the homogenate was centrifuged to give the supernatant. Trifluoroaceticacid (TFA) was added to the supernatant in 0.05% and the mixture wasapplied to C18 Column (Prep C18 125 Å; Waters). The peptide bound to thecolumn was stepwise eluted with 10%, 40% and 60% acetonitrile containing0.5% TFA. A 2-fold volume of 20 mM ammonium acetate (pH 4.7) was addedto the 40% acetonitrile fraction for dilution. The mixture was appliedto ion exchange column HiPrep CM-Sepharose FF (Pharmacia). The peptidebound to the ion exchange column was eluted on a concentration gradientof 0 to 0.5 M NaCl in 20 mM ammonium acetate (pH 4.7) containing 10%acetonitrile. A 2-fold volume of cold acetone was added to the NaClfraction (0.3 to 0.35 M) containing the active substance mostabundantly. The precipitates were removed by centrifugation and thesupernatant was concentrated through an evaporator. TFA was added to theconcentrated supernatant in 0.1%. The mixture was applied to reversephase HPLC column RESOURCE RPC (Pharmacia) to effect further separation.The separation from RESOURCE RPC was performed on a concentrationgradient of 20 to 30% acetonitrile, whereby the main activity was elutedon about 22% acetonitrile. A 3-fold volume of cold acetone was added tothe active fraction. The precipitates were removed by centrifugation andthe supernatant was concentrated through the evaporator. TFA was addedto the concentrated supernatant in 0.1%. The mixture was applied toreverse phase HPLC column Vydac C18 218TP5415 (Vydac) to effect furtherseparation. The separation from Vydac C18 218TP5415 was performed on aconcentration gradient of 20 to 30% acetonitrile, whereby the mainactivity was eluted on about 25% acetonitrile. The active fraction wasseparated through cation exchange column TSK-gel CM-2SW (Toso Co., Ltd.)on a concentration gradient of 0.3 to 0.5 M NaCl in 20 mM ammoniumacetate (pH 4.7) containing 10% acetonitrile, whereby the main activitywas eluted on about 0.5 M NaCl. TFA was added to the activity-containingfraction from CM-2SW column in 0.1%. The final purification was madethrough reverse phase HPLC column μRPC C2/C18 SC2.1/10 on aconcentration gradient of 16 to 24% acetonitrile. Thus, a single peakwhich coincided with the activity was obtained (FIG. 13).

Example 11

[1268] N-Terminal Amino Acid Sequencing of the Finally Purified Productand Determination of its Molecular Weight by Mass Spectrum

[1269] With respect to the finally purified product obtained in EXAMPLE10, approximately a half was analyzed with a protein sequencer (model491cLC; Applied Biosystems) for the N-terminal amino acid and the otherhalf was analyzed by ESIMS (Thermoquest).

[1270] As a result of the N-terminal sequencing, the sequencecorresponding to the positions 26 to 49 of bovine GPR7 ligand precursorcould be read in cycles 2 to 25 (FIG. 14). Since cycle 1 could not beidentified (x), the product was presumed to undergo a post-translationalmodification. The sequence after cycle 2 was identified clearly to bethe sequence described above.

[1271] In ESIMS (FIG. 15, upper column), the value of 3241.5 wasobtained in a full mass scan mode. Based on the molecular weightcalculated from the mass spectrum and the analysis results of MS/MSspectrum (FIG. 15, lower column), it was presumed that either one of theN-terminal two residues would undergo a post-translational modification.Taking into account the N-terminal sequencing results together, it waspresumed that Trp at position 1 would be modified.

[1272] Putting the isotonic profile (FIG. 16) of trivalent molecularions determined in a zoom scan mode together, the substance was presumedto be a GPR7 ligand of 29 residues, which would be brominated on thetryptophan residue at the 1-position.

[1273] To confirm the presumption, PTH standard was prepared fromDL-5-bromotryptophan (Aldrich) and DL-6-bromotryptophan (Biosynth),followed by sequencing.

[1274] To 200 nmols of DL-5-bromotryptophan or DL-6-bromotryptophan, 20μl of ethanol:triethylamine:DW:phenyl isothiocyanate (sigma)=7:1:1:1 wasadded. The mixture was reacted at room temperature for 20 minutes. Afterdrying, 50 μl of TFA was added thereto. The mixture was reacted at 50°C. for 10 minutes. After during, 50 μl of HCl: methanol=1:1 was addedthereto, followed by reacting at 50° C. for 10 minutes. The reactionmixture was purified on reverse phase HPLC to give the PTH derivative of5-bromotryptophan or 6-bromotryptophan. The final product was identifiedon a protein sequencer (FIGS. 17 and 18). These PTH derivatives weremixed with 20 amino acid PTH standard (ABI), and a protocol was preparedto separate the derivatives (TABLES 1 and 2). When the endogenous bovineGPR7 ligand was analyzed, the amino acid at the 1-position coincidedwith the peak of PTH-6-bromotryptophan (FIG. 19).

[1275] Based on the results of analysis, the finally purified productfrom bovine hypothalamus was found to be a peptide of 29 amino acids(SEQ ID NO:67) corresponding to the 25th Trp to. 53rd Ala of bovine GPR7ligand precursor, in which Trp at position 1 was 6-brominated by apost-translational modification. TABLE 1 Cycle # Cartridge cycle Flaskcycle Gradient Pulsed-Liquid cLC: Default Cart-PL 6mmGFF cLC FlaskNormal cLC Normal 1 cLC 1 None Prepare Pump cLC Prepare Pump cLC 2 NoneFlask Blank cLC Normal 1 cLC 3 Cart Begin cLC Flask Standard cLC Normal1 cLC BrTrp-liquid cLC: Default Cart-PL 6mmGFF cLC Flask Normal cLCNormal for BrW cLC 1 Sample wash Prepare Pump cLC Prepare Pump cLC 2None Flask Blank cLC BrTrp cLC 3 Cart Begin cLC Flask Standard cLC BrTrpcLC 4 Cart-PL 6mmGFF cLC Flask Normal cLC BrTrp cLC 5 Cart-PL 6mmGFF cLCFlask Normal cLC BrTrp cLC

[1276] TABLE 1 shows a comparison in cycle and gradient between a methodfor normal peptide (Pulsed-Liquid cLC) and a method for bromotryptophan(BrTrp-liquid cLC). TABLE 2 Time (min) 0 0.4 4 22 22.6 29 33 Normal 1 %B 8 10 20 47 90 90 70 cLC Normal % B 8 10 20 44 90 90 70 for BrW cLC 0.00.4 4 22.0 28.0 28.6 32.0 33.0 BrTrp cLC % B 8 10 20 44 44 90 90 70

[1277] TABLE 2 shows a comparison in gradients prepared for the analysisof a normal peptide (Normal 1 cLC) and bromotryptophan (Normal for BrWcLC, BrTrp cLC), on 491cLC protein sequencer (ABI).

[1278] The N-terminal sequencing was performed on 491cLC proteinsequencer (ABI) by the analysis method for normal peptide (Normal 1cLC)with a modification (BrTrp-liquid cLC) for bromotryptophan analysis.Other conditions than those described above were set as instructed inthe manual provided by the manufacturer. When a modified gradient (BrTrpcLC) is used, 5- and 6-bromotryptophans, which have different positionsfor Br added, can be discriminated from each other.

[1279] When the analysis is made by the modified method or BrTrp-liquidcLC, the gradient for analysis of 5-/6-bromotryptophan is adapted onlyto blank, standard and up to cycle 2, and a different gradient (Normalfor BrW cLC) is adapted to and after cycle 3.

Example 12

[1280] Acquisition of Bovine GPR7 Ligand Precursor Gene from BovineHypothalamus cDNA by PCR

[1281] Using bovine hypothalamus cDNA as a template, PCR was performedfor amplification, using two synthetic DNAs below. BF1:5′-CCCATGGCCGGGCCCGCGATGCTGG (SEQ ID NO:82) TCGCC-3′ BR1:5′-TCACTTGCGACAGTCCGAGGCGCTG (SEQ ID NO:83) AGCGA-3′′

[1282] The reaction solution for PCR contained 1 μl of cDNA solution,0.5 μl of BF1 (10 μM), 0.5 μl of BF2 (10 μM), 2.5 μl of 10× reactionsolution attached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq(CLONTECH, Inc.), to which 17.5 μl of Otsuka distilled water was addedto make the total volume 25 μl. The reaction solution was applied to PCRusing Thermal Cycler 9600. The conditions for PCR were set forth: afterdenaturation at 95° C. for 2 minutes, the cycle set to include 98° C.for 10 seconds, 60° C. for 20 seconds and 72° C. for 20 seconds wasrepeated 35 times. After it was confirmed by electrophoresis using analiquot of the PCR product that the PCR product of about 400 bp wasamplified, the PCR product was purified using Quiagen PCR purificationKit and directly sequenced to obtain the sequence shown by FIG. 20. Theamino acid sequence deduced from the DNA sequence of FIG. 20 was thesequence shown in FIG. 21. Next, the PCR product recovered from the gelwas subcloned to Escherichia coli JM109 using TA Cloning Kit(Invitrogen, Inc.) to acquire Escherichia coli JM109/pTAbGPR7L-1.Plasmid pTAbGPR7L-1 was extracted from Escherichia coli obtained by thesubcloning, using a plasmid extractor (Kurabo Co., Ltd.) to identify thebase sequence of the inserted fragment. It was confirmed that thesequence was the same as that of bovine GPR7 ligand cDNA.

Example 13

[1283] Synthesis of GPR7 Ligand

[1284] GPR7 ligand (GPR7L) and GPR8 ligand (GPR8L) were synthesized bythe Fmoc/DCC/HOBt protocol, using ABI 433 peptide synthesizer.DL-6-Bromotryptophan (Biosynth) was changed toBoc-DL-6-bromotryptophan-OMe and then subjected to chiral resolution,which was used for peptide synthesis, respectively. (1) DTrp (6Br)1-human GPR7L (29) (wherein N-terminal D-tryptophan in the amino acidsequence represented by SEQ ID NO:4 was brominated at the 6-position):(D-Trp(6Br)-Tyr-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Leu-Arg-Arg-Ser-Pro-Tyr-Ala) (2) LTrp (6Br)1-human GPR7L(29) (wherein N-terminal L-tryptophan in the amino acid sequencerepresented by SEQ ID NO:4):(Trp(6Br)-Tyr-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Leu-Arg-Arg-Ser-Pro-Tyr-Ala) (3) Trp1-human GPR7L (29)(SEQ ID NO:4):(Trp-Tyr-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Leu-Arg-Arg-Ser-Pro-Tyr-Ala) (4) Trp1-humanGPR7L (23) (SEQID NO:1):(Trp-Tyr-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Leu) (5) DTrp(6Br)1-bovine GPR7L (29) (wherein N-terminalD-tryptophan in the amino acid sequence represented by SEQ ID NO:67 wasbrominated at the 6-position):(D-Trp(6Br)-Tyr-Lys-Pro-Thr-Ala-Gly-Gln-Gly-Tyr-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Phe-His-Arg-Ser-Pro-Tyr-Ala) (6) LTrp(6Br)1-bovineGPR7L (29) (wherein N-terminal L-tryptophan in the amino acid sequencerepresented by SEQ ID NO:67 was brominated at the 6-position):(Trp(6Br)-Tyr-Lys-Pro-Thr-Ala-Gly-Gln-Gly-Tyr-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Phe-His-Arg-Ser-Pro-Tyr-Ala) (7) Trp1-bovine GPR7L (29)(SEQ ID NO:67):(Trp-Tyr-Lys-Pro-Thr-Ala-Gly-Gln-Gly-Tyr-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Phe-His-Arg-Ser-Pro-Tyr-Ala) (8) Trp1-rat GPR7L (29) (SEQ IDNO:6):(Trp-Tyr-Lys-Pro-Ala-Ala-Gly-Ser-HisI-His-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Ser-Phe-His-Arg-Phe-Pro-Ser-Thr) (9) Trp1-rat GPR7L (24) (SEQ IDNO:3):(Trp-Tyr-Lys-Pro-Ala-Ala-Gly-Ser-His-His-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Ser-Phe-His) (10) Trp1-human GPR8L (23) (SEQ ID NO: 100):(Trp-Tyr-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu) (WO 01/98494)

Example 14

[1285] Effect of GPR7 Ligand on Feed Uptake in Rat by LateralVentricular Injection

[1286] The effect of GPR7 ligand (GPR7L) on feed uptake in rat bylateral ventricular injection was examined. Rat was caged at roomtemperature of 25° C. while lighting for 8 to 20 o'clock. Mature Wistarmale rats (300-320 g body weight upon surgery) were anesthetized with anintraperitoneal injection of 50 mg/kg pentobarbital and placed in a ratbrain stereotaxic instrument. The level of incisor bar was 3.3 mm belowthe interaural line. The skull was exposed, and using a dental drill ahole was made on the bone for implantation of guide cannula AG-8 (innerdiameter of 0.4 mm, outer diameter of 0.5 mm, EICOM Corporation). Inaddition, an anchor screw was buried at 3 positions around the hole. Astainless-steel guide cannula, AG-8, was inserted in such a manner thatthe tip was situated at the upper part of the lateral ventricle.Stereotaxic coordinates were taken from the atlas according to the atlasof Paxinos & Watson (1986) from bregma AP: −0.8 mm, L:1.5 mm and H:−4.5mm. The guide cannula was secured to the skull using a dental cement andan anchor screw. A stainless-steel dummy cannula AD-8 (outer diameter of0.35 mm, EICOM Corporation) was then passed through the guide cannulaand locked in position with a cap nut (EICOM Corporation). After thesurgery, rats were housed in individual cages.

[1287] After the guide cannula was implanted, rats were caged for abouta week to recover from surgical operation. The cap nut and dummy cannulainserted into the rat skull were disconnected and instead, astainless-steel microinjection cannula AMI-9 (inner diameter of 0.17 mm,outer diameter of 0.35 mm, EICOM Corporation) connected to a Teflon(registered trademark) tube (length of 50 cm, inner diameter of 0.1 mm,outer diameter of 0.35 mm, EICOM Corporation) was inserted into theskull. The length of the microinjection cannula was adjusted beforehandto expose the tip from the guide cannula by 1 mm. One end of the Teflon(registered trademark) tube was connected to a microsyringe pump andeither sterile distilled water (Otsuka Pharmaceutical Co., Ltd.) ornon-brominated GPR7L (Trp1-bovine GPR7L (29), SEQ ID NO:67) synthesizedin EXAMPLE 13 dissolved in distilled-water was infused, in a totalvolume of 10 μl (10 nmols/rat), into the lateral ventricle at a flowrate of 5 μl/min. After a 2 minute standby time following the infusion,the microinjection cannula was disconnected and the dummy cannula waslocked in position again with a cap nut. The infusion was continued from19:00 to 20:00 o'clock, and the feed uptake thereafter was measured withpassage of time, using a feed uptake measuring device Feed-Scale(Columbus, Inc.). As shown in FIG. 22, a significant increase in feeduptake from 2 hours after the administration was noted in the groupadministered with non-brominated GPR7L, as compared to the controlgroup.

Example 15

[1288] Assay for Bovine Endogenous GPR7 Ligand by FTMS

[1289] Bovine endogenous GPR7 was analyzed on Apex II (Burker Daltonics)by ESIFTMS (FIG. 23, upper). In FIG. 23, [M+5H]⁵⁺ ions are shown in anenlarged drawing and the [M+5H]⁵⁺ ion isotope theoretical profile of1Br-added bovine GPR7L is shown at the lower column. The modifiedproduct was identified to be Br, since the isotope profile and massspectral data matched well.

Example 16

[1290] Study of Tissue Distribution of GPR7 Ligand mRNA in Human byRT-PCR

[1291] The expression level of mRNA was assayed in a manner similar toEXAMPLE 9, except that cDNAs used as templates were prepared from polyA+RNA (CLONTECH) derived from various human organs by the followingprocedures. Using reverse transcriptase or SuperScript II as a randomprimer and reverse transcriptase, cDNA was prepared from 1 μg of RNA andthe reaction was carried out at 42° C. in accordance with theinstruction manual attached. After completion of the reaction, theprecipitates in ethanol were dissolved in 100 μl. Also, the expressionlevel was quantified in a manner similar to EXAMPLE 9 using SequenceDetection System Prism 7700, except that the following were used foramplification and detection: 5′-CGCTCCCAGCCCTACAGA-3′ (SEQ ID NO:90) and5′-TCGCCTTGCACTGGTAGGTC-3′ (SEQ ID NO:91) as primers and as TaqManprobe, 5′-(Fam) AGCCTCGCTGTGTGCGTCCAGGAC-(Tamra)-3′ (SEQ ID NO:92).

[1292] The expression level of GPR7 mRNA in various human tissuesobtained was assessed in terms of a copy number per 1 ng of poly(A)⁺ RNA(FIG. 24).

Example 17

[1293] Study of Tissue Distribution of Rat GPR7 (rat TGR26) mRNA byRT-PCR

[1294] The expression level of mRNA was assayed in a manner similar toEXAMPLE 9. Using the cDNAs derived from various organs of rat used inEXAMPLE 9, the expression level of rat GPR7 mRNA was determined, exceptthat except that the following were used for amplification anddetection: 5′-TGCGTGCTATCCAGCTAGACAG-3′ (SEQ ID NO:93) and5′-AGAGGAGGCACACAGCCAGAAT-3′ (SEQ ID NO:94) as primers and5′-(Fam)CGTGCCAAGAAACGCGTGACCTTGTT-(Tamra)-3′ (SEQ ID NO:95) as TaqManprobe.

[1295] The expression level of GPR7 mRNA in various tissues of ratobtained was assessed in terms of a copy number per 1 ng of poly(A)+ RNA(FIG. 25).

Example 18

[1296] Acquisition of Bovine GPR7 Gene from Bovine Hypothalamus cDNA byPCR

[1297] Using bovine hypothalamus cDNA as a template, amplification wasperformed by PCR using the following two synthetic DNAs. BGPR7F:5′-GTCGACCGAGTGTCTGTCCTCGCCAGGATG-3′ (SEQ ID NO:96) BGPR7R:5′-GCTAGCTCCTTGTTATCGGGCTCAGGAGGTGGT-3′ (SEQ ID NO:97)

[1298] The reaction solution for PCR contained 1 μl of cDNA solution,0.5 μl of BGPR7F (10 μM), 0.5 μl of BGPR7R (10 μM), 2.5 μl of 10×reaction solution attached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq(CLONTECH, Inc.), to which 17.5 μl of Otsuka distilled water was addedto make the total volume 25 μl. The reaction solution was applied to PCRusing Thermal Cycler 9600. The conditions for PCR were set forth: afterdenaturation at 95° C. for 2 minutes, the cycle set to include 98° C.for 10 seconds, 60° C. for 20 seconds and 72° C. for 60 seconds wasrepeated 40 times. It was confirmed by electrophoresis using an aliquotof the PCR product that the PCR product of about 1000 bp was amplified.The PCR product was then purified using Quiagen PCR purification Kit(QIAGEN, Inc.) and directly sequenced to obtain the sequence shown byFIG. 26. The amino acid sequence deduced from the DNA sequence of FIG.26 was the sequence shown in FIG. 27. Next, the PCR product recoveredfrom the gel was subcloned to Escherichia coli JM109, using TA CloningKit (Invitrogen, Inc.), to acquire Escherichia coli JM109/pTAbGPR7.Plasmid pTAbGPR7 was extracted from Escherichia coli obtained by thesubcloning, using a plasmid extractor (Kurabo Co., Ltd.) to identify thebase sequence of the inserted fragment. It was confirmed that thesequence was the same as that of bovine GPR7 receptor cDNA.

Example 19

[1299] Acquisition of Bovine GPR8 Gene from Bovine Hypothalamus cDNA byPCR

[1300] Using bovine hypothalamus cDNA as a template, amplification wasperformed by PCR using the following two synthetic DNAs. BGPR8F:5′-GTCGACCATGATGGAGGCCACTGGGCTGGAAGG-3′ (SEQ ID NO:98) BGPR8R:5′-GCTAGCTTATGCCCCCTGGCACCGACATGCGGT-3′ (SEQ ID NO:99)

[1301] PCR was carried out in a manner similar to EXAMPLE 18. The PCRproduct obtained was purified using Quiagen PCR purification Kit anddirectly sequenced to obtain the sequence shown by FIG. 28. The aminoacid sequence deduced from the DNA sequence of FIG. 28 was the sequenceshown in FIG. 29. Next, the PCR product recovered from the gel wassubcloned to Escherichia coli JM109, using TA Cloning Kit (Invitrogen,Inc.), to acquire Escherichia coli JM109/pTAbGPR8. Plasmid pTAbGPR8 wasextracted from Escherichia coli obtained by the subcloning, using aplasmid extractor to identify the base sequence of the insertedfragment. It was confirmed that the sequence was the same as that ofbovine GPR8 cDNA.

Example 20

[1302] Purification of GPR7 Ligand from the Culture Supernatant of HumanGPR7 Ligand-Expressed CHO Cells

[1303] The culture supernatant of human GPR7 ligand-expressed CHO cellsconstructed in EXAMPLE 5 was collected to make the volume 2 liters andstored at −80° C. The culture supernatant was thawed, boiled in hotwater and then centrifuged to obtain the supernatant. Trifluoroaceticacid (TFA) was added in 0.05% to the supernatant and the mixture wasapplied to C18 Column (Prep C18 125 Å; Waters, Inc.). The peptide boundto the column was stepwise eluted with 10, 40 and 60% acetonitrilecontaining 0.5% TFA. The 30% acetonitrile fraction was diluted with a3-fold volume of 20 mM ammonium acetate (pH 4.7) and the dilution wasapplied to ion exchange column HiPrep CM-Sepharose FF (Pharmacia). Thepeptide bound to the column was eluted in a concentration gradient of 0M to 0.5 M NaCl in 20 mM ammonium acetate (pH 4.7) containing 10%acetonitrile. An aliquot of each fraction was desalted using Sep-Pakplus C18 Cartridge (Waters, Inc.), the intracellular cAMP productionsuppression activity specific to human GPR7-expressed CHO cells wasassayed. TFA was added in 0.1% to the CM-Sepharose fraction found tohave a specific activity to human GPR7-expressed CHO cells, which wasseparated by passing through reverse phase HPLC column RESOURCE RPC(Pharmacia). The separation through RESOURCE RPC was carried out in aconcentration gradient of 15-30% acetonitrile. The main intracellularcAMP production suppression activity specific to the humanGPR7-expressed CHO cells was eluted on about 22% acetonitrile. Thisactive fraction was separated by passing through cationic ion exchangecolumn TSK gel CM-SW (Toso Co., Ltd.) in a concentration gradient of0.2-0.5 M NaCl in 20 mM ammonium acetate (pH 4.7) containing 10%acetonitrile. The main intracellular cAMP production suppressionactivity was eluted on about 0.3 M NaCl. TFA was added in 0.1% to thefraction from the CM-2SW column. Final purification of the mixture onreverse phase column μRPC C2/C18 SC2.1/10 gave a single peak, whichcoincided with the intracellular cAMP production suppression activityspecific to the human GPR7-expressed CHO cells (FIG. 30).

[1304] Analysis of the N-terminal amino acids in the finally purifiedproduct using a protein sequencer (model 492; Applied Biosystems, Inc.)gave the amino acid sequence shown in FIG. 31.

[1305] Also, the molecular weight of the finally purified product wasdetermined using ESI-MS (Thermoquest, Inc.) and found to be 2505.6 (FIG.32).

[1306] Based on these analytical results, the finally purified productwas found to be a peptide of 24 amino acids corresponding to Trp25 toArg48 in the precursor.

Example 21

[1307] Preparation of Iodine-Labeled Human GPR7 Ligand

[1308] A mixture of 20 μl of hGPR7L-23 (SEQ ID NO:1) (0.1 mM or 1 mM),20 μl of lactoperoxidase (Sigma; prepared using 10 μg/ml and 0.1MHEPES-NaOH pH7.0), 20 μl of Idoine-125 (manufactured by Amersham, MS-30,74 MBq), 20 μl of 0.005% hydrogen peroxide (manufactured by Wako PureChemical Industries, Ltd.) was allowed to stand at room temperature for20 to 30 minutes. Then, 600 μl of 0.1% TFA was added to the mixture. Themixture was applied to reverse phase HPLC for separation and the peaksof the two'labeled products were fractionated in a tube charged with 1mL of DMSO. Immediately, the fraction was stored on ice and an aliquotwas provided for measurement of the radioactivity with a γ-counter. Theremaining preparation was dispensed and stored at −30° C.

Example 22

[1309] Preparation of Human GPR7-Expressed CHO Cell Membrane Fraction

[1310] Human GPR7-expressed CHO cells were cultured in a flask. Theflask was washed with 5 mM EDTA/PBS to strip the cells off. The cellswere stripped off with 5 mM EDTA/PBS and centrifuged for recovery. Therecovered cells were suspended in 25 mL of a buffer for preparingmembrane fraction (50 mM Tris-HCl, pH7.5, 5 mM EDTA, 0.1% bovine serumalbumin (manufactured by Sigma), 0.5 mM PMSF (manufactured by Wako PureChemical Industries, Ltd.), 20 μg/mL leupeptin (manufactured by PeptideResearch Institute), 0.1 μg/mL pepstatin A (manufactured by PeptideResearch Institute) and 4 μg/mL E-64 (manufactured by Peptide ResearchInstitute)), followed by homogenization on ice using a polytron (12,000rpm, 15 seconds×3 times). The homogenate was centrifuged at 4° C. under1,000 g for 10 minutes with a high speed cooling centrifuge to recoverthe supernatant. After 25 mL of the buffer for preparing membranefraction was added to the precipitates, the supernatant was recovered bythe same procedures. These supernatants were combined, applied to a cellstrainer, dispensed in a super centrifuge tube and centrifuged at 4° C.under 100,000 g for an hour. The pellets were recovered and suspended ina small quantity of the buffer for preparing membrane fractions. Afterfurther suspending with a Teflon (registered trademark) homogenizer, analiquot of the suspension was used to determine a protein level. Theremaining suspension was dispensed and stored at −80° C.

Example 23

[1311] Scatchard Analysis Using Human GPR7-Expressed CHO Cell MembraneFraction

[1312] Scatchard analysis was performed using human GPR7-expressed CHOcell membrane fraction and [Tyr (¹²⁵I)¹¹]-hGPR7L-23 (SEQ ID NO:1). Themembrane fraction was diluted in a final concentration of 1 μg/well withan assay buffer (50 mM Tris-HCl, pH7.5, 5 mM EDTA, 0.1% bovine serumalbumin (manufactured by Sigma), 0.5 mM PMSF (manufactured by Wako PureChemical Industries, Ltd.), 20 μg/mL leupeptin (manufactured by PeptideResearch Institute), 0.1 μg/mL pepstatin A (manufactured by PeptideResearch Institute) and 4 μg/mL E-64 (manufactured by Peptide ResearchInstitute)), and the labeled product was diluted in 400 μM, 200 μM, 100μM, 50 μM, 25 μM and 10 μM. Using a polyproprene-made 96-well plate, 50μl each of the assay buffer alone (total) and hGPR7L-23 (NSB) in a finalconcentration of 2 μM were dispensed in each well. To each well, 25 μlof the solution of the labeled product was added. After agitation, 25 μlof the diluted membrane fraction was added/mixed, followed by incubationat room temperature for 1.5 hour. Using a cell harvester for the 96-wellplate, adsorption was made onto a filter unit (GF/C, treated withpolyethyleneimine), which had previously been made wet with an assaybuffer (50 mM Tris-HCl, pH7.5). After washing 5 times with the assaybuffer, the filter unit was thoroughly dried. As an input, the dilutionof the labeled product was directly added to a filter unit (GF/C,treated with polyethyleneimine) and dried. After 50 μl of a liquidscintillator was dispensed thereto, the radioactivity was counted on aTop Count (Packard) and the data was analyzed in triplet (FIG. 33),thereby to obtain the values of Bmax=1.28 pmol/mg protein and Kd=35.5μM.

Example 24

[1313] Test on Binding Inhibition of Various Peptides Against HumanGPR7-Expressed CHO Cells

[1314] Using the assay buffer, the human GPR7-expressed CHO cellmembrane fraction was diluted in a final concentration of 1 μg/well andiodine-labeled hGPR7L-23 (SEQ ID NO:1) was diluted in a finalconcentration of 100 μM. The peptides shown in TABLE 3 are thoseobtained by diluting the stock solution of 10 M or 10⁻³ M with the assaybuffer in 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M and 10⁻¹¹ M.As NBS, hGPR7L-23 was prepared in a final concentration of 10⁻⁵ M. Thesample solution and NSB prepared were dispensed on a polypropylene-made96-well plate, the assay buffer was dispensed thereto to make the totalvolume 50 μl, and 25 μl of the iodine-labeled product dilution was addedthereto. After agitation, 25 μl of the solution of human GPR7-expressedCHO cell membrane fraction were dispensed thereto and agitated, followedby incubation at room temperature for 1.5 hour. Using a cell harvesterfor the 96-well plate, the culture medium was adsorbed onto a filterunit, which had previously been wetted with an assay buffer (50 mMTris-HCl, pH7.5). After washing 5 times with the assay buffer, thefilter unit was thoroughly dried. After 50 μl of a liquid scintillatorwas dispensed thereto, the radioactivity was counted on a Top Count(Packard) and the data was analyzed in triplet (FIG. 33). The resultsobtained by the test on binding inhibition of various peptides againsthuman GPR7-expressed CHO cell membrane fraction are shown in terms ofIC₅₀ values in TABLE 3. TABLE 3 Peptide IC50(nM) DTrp(6Br)1-humanGPR7L(29) 13 LTrp(6Br)1-human GPR7L(29) 0.32 Trp1-human GPR7L(29) 0.33Trp1-human GPR7L(23) 1.6 Trp1-human GPR8L(23) 0.4 DTrp(6Br)1-bovineGPR7L(29) 6.1 LTrp(6Br)1-bovine GPR7L(29) 0.34 Trp1-bovine GPR7L(29)0.31 Trp1-rat GPR7L(29) 0.34 Trp1-rat GPR7L(24) 0.30

Example 25

[1315] Comparison in Agonist Activity of Various Peptides on GPR7 andGPR8-Expressed CHO Cells

[1316] The intracellular cAMP production suppression activity ofpeptides associated with various GPR7 ligands on CHO cells, in whichhuman GPR7, bovine GPR8, human GPR8, human GPR8, bovine GPR8 and ratGPR7 were expressed, was examined. Each of the receptor-expressed cellswas passaged on a 96-well plate in 4×10⁴ cells/well, followed byincubation for 1 day at 37° C. under 5% CO₂ and 95% air. An assay bufferwas prepared by adding 20 mM HEPES, pH7.4, 0.1% bovine serum albumin and0.2 mM 3-isobutyl-1-methylxanthine (Sigma) to Hanks' Balanced SaltSolution (Gibco BRL). The plate incubated overnight was first washedtwice with 150 μl of the assay buffer, and then exchanged with 150 μl ofthe assay buffer, followed by incubation for 30 minutes at 37° C. in100% air. By adding 4 μM forskolin to the assay buffer, a buffer forsample dilution was prepared and the stock solution (10⁻² M of 10⁻³ M)was diluted with the buffer thus obtained to prepare sample solutions infinal concentrations of 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M and 10⁻¹⁰ M. Theplate incubated with the assay buffer for 30 minutes was taken out.After washing twice with the assay buffer, 50 μl of the assay buffer andthen 50 μl of the sample solution were added thereto. Each sample wasassayed in triplet. Furthermore, the assay buffer of the same volume forassaying the basal level and a buffer supplemented with forskolin forassaying the maximum level were added. After the plate was incubated for30 minutes at 37° C. in 100% air, the intracellular cAMP level wasassayed using cAMP-Screen™ System (ABI, Inc.) according to the protocolattached to the kit. A difference between the maximum cAMP level and thecAMP level when each sample was added was calculated and the percentageof cAMP production level promoted by forskolin was worked out, which wasmade the cAMP production inhibition rate. The IC₅₀ values of respectivesamples are shown in TABLE 4. TABLE 4 IC50(nM)

human GPR7 human GPR8 bovine GPR7 bovine GPR8 rat GPR7 DTrp(6Br)1-humanGPR7L(29) 106 770 360 424 49 LTrp(6Br)1-human GPR7L(29) 0.44 32 1.8 510.47 Trp1-human GPR7L(29) 0.45 49 2.6 52 0.29 Trp1-human GPR7L(23) 0.5844 3.5 47 0.28 Trp1-human GPR8L(23) 0.82 3.7 2.4 4.8 0.32DTrp(6Br)1-bovine GPR7L(29) 35 198 77 34 2.5 LTrp(6Br)1-bovine GPR7L(29)0.58 9.7 1.9 4.0 0.21 Trp1-bovine GPR7L(29) 0.43 8.1 1.2 5.4 0.25Trp1-rat GPR7L(29) 0.86 8.8 1.2 14 0.30 Trp1-rat GPR7L(24) 0.31 2.8 0.513.5 0.18

Example 9

[1317] Study of Expression Distribution of GPR7 Ligand mRNA in Rat Brainby In Situ Hybridization

[1318] Wistar rat was laparotomized under anesthesia with Nembutal and250 ml of 0.9% aqueous sodium chloride solution was infused through theleft ventricle and then with 250 ml of 4% p-formaldehyde solution. Afterthe brain withdrawn was immersed in the solution for 4 hours at 4° C.,the solution was replaced by 20% sucrose solution. The brain wasimmersed for further 3 days at 4° C. to obtain the brain sample providedfor analysis.

[1319] GPR7 ligand, antisense and sense probe were prepared by thefollowing method.

[1320] First, rat GPR7 ligand cDNA was inserted into plasmid vectorpBluescript II KS+ (Stratagene) by publicly known methods. This plasmidwas inactivated by treating with restriction enzyme BamHI or XbaI, whichwas dissolved in TE, respectively, in 0.52 μg/ml and 0.47 μg/ml. To 2 μlof the BamHI-treated product, 40 U of T3 RNA polymerase (Roche), 2 μl ofthe supplied 10× buffer, 20U of RNase inhibitor (Roche) and 2 μl of DIGRNA Labeling Mix, 10× (Roche), water was added to make the final volume20 μl. After the mixture was reacted at 37° C. for 2 hours, the reactionwas terminated by adding 2 μl of 0.2 M EDTA, and the riboprobe formed byethanol precipitation was recovered and used as an antisense probe.Also, to 2 μl of the XbaI-treated product, 40 U of T3 RNA polymerase(Roche), 2 μl of the supplied 10× buffer, 20U of RNase inhibitor (Roche)and 2 μl of DIG RNA Labelling Mix, 10× (Roche), water was added to makethe final volume 20 μl. The mixture was reacted at 37° C. for 2 hours.After the reaction was terminated by adding 2 μl of 0.2 M EDTA, theriboprobe formed by ethanol precipitation was recovered and used as asense probe. The respective concentrations were measured and the probeswere dissolved in RNase free water to set the concentrations ofantisense and sense probes at 0.29 μg/ml and 0.27 μg/ml, respectively.

[1321] In situ hybridization was carried out by the following method.The brain sample prepared as described above was sliced on CryostatCM3050 (Leica) at 25 μm thick in the frontal plane. The slice formed waswashed twice with 10 ml of 4×SSC for 5 minutes. Then, Protenase K(Sigma) was added to 10 ml of PK buffer (pH7.4, 10 mM Tris-HCl, 10 mMEDTA) in a final concentration of 2.5 mg/ml, followed by reacting themat 37° C. for 10 minutes. The reaction mixture was washed twice with 10ml of 4×SSC for 5 minutes. Acetic anhydride was added to 10 ml of anacetylation buffer (pH 7.5, 100 mM triethanolamine) in 0.25%, followedby reacting at room temperature for 10 minutes. The mixture was washedwith 10 ml of 4×SSC for 5 minutes. After this operation was repeatedtwice, the slice was added to 1 ml of a hybrid buffer (pH 7.4, 60%formamide, 10 mM Tris-HCl, 200 μg/ml yeast t-RNA, 1× Denhardt's reagent,10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA) supplementedwith 0.2 μg/ml each of the antisense and the sense probe. Hybridizationwas performed at 55° C. for 13 hours, followed by washing at 55° C. for15 minutes twice with 10 ml of a wash buffer (2×SSC, 50% formamide).RNaseA (Sigma) was added to RNase buffer (pH8.0, 10 mM Tris-HCl, 1 mMEDTA, 0.5M NaCl) in 2.5 μg/ml and the slice was transferred theretofollowed by reacting them at 37° C. for 30 minutes. The reaction mixturewas washed at 55° C. for 15 minutes with 10 ml of the wash buffer. Afterthis operation was repeated twice, the mixture was washed with 0.4×SSCat 55° C. for 15 minutes. The slice was transferred to a solutionmixture of 0.1 g of a blocking solution (Roche) and Buffer A (pH 7.5,100 mM Tris-HCl, 150 mM NaCl) to react them at room temperature for anhour. The slice was transferred to 1 ml of Buffer A containing TritonX-100 and supplemented with 0;75U of anti-digoxygenin-AP, Fab fragments(Roche), followed by reacting them at 4° C. for 16 hours. The reactionmixture was then washed with 10 ml of Buffer A at room temperature for15 minutes. After this operation was repeated twice, the mixture waswashed with 10 ml of Buffer B (pH 9.5, 100 mM Tris-HCl, 100 mM NaCl, and50 mM MgCl₂) at room temperature for 15 minutes. To 10 ml of Buffer B,40 oil of NBT solution (Roche) and 30 μl of X-phosphate solution wereadded, and the slice was transferred to the mixture to perform acolor-forming reaction. After reacting at room temperature for 24 hours,the slice was transferred to 50 ml of TE. The slice was applied ontoMAS-coated slide glass (Matsunami Glass Ind. Ltd.). After air-drying, acover glass was applied thereon with a sealant (50% glycerol, 5%gelatin). The areas where the color was developed specifically to theantisense probe were the medial and lateral preoptic areas ofhypothalamus, the lateral hypothalamic field, the CA1-CA3 areas ofhippocampal pyramidal cells, the mesencephalic aqueduct ventral divisionof the midbrain, etc. In these areas, any color formation was notdetected by the sense probe.

INDUSTRIAL APPLICABILITY

[1322] The peptide of the present invention and its DNA, the bovine GPR7of the present invention and its DNA, as well as the bovine GPR8 of thepresent invention and its DNA are useful as pharmaceuticals for theprevention/treatment of, e.g., anorexia, appetite (eating) stimulants,etc.

[1323] In addition, the peptide, etc. of the present invention are alsouseful for screening GPR7 agonists or antagonists, etc.

1 108 1 23 PRT Homo sapiens 1 Trp Tyr Lys Pro Ala Ala Gly His Ser SerTyr Ser Val Gly Arg Ala 5 10 15 Ala Gly Leu Leu Ser Gly Leu 20 2 24 PRTMus sp. 2 Trp Tyr Lys Pro Ala Ala Gly Pro His His Tyr Ser Val Gly ArgAla 5 10 15 Ser Gly Leu Leu Ser Ser Phe His 20 3 24 PRT Rattus sp. 3 TrpTyr Lys Pro Ala Ala Gly Ser His His Tyr Ser Val Gly Arg Ala 5 10 15 AlaGly Leu Leu Ser Ser Phe His 20 4 29 PRT Homo sapiens 4 Trp Tyr Lys ProAla Ala Gly His Ser Ser Tyr Ser Val Gly Arg Ala 5 10 15 Ala Gly Leu LeuSer Gly Leu Arg Arg Ser Pro Tyr Ala 20 25 5 29 PRT Mus sp. 5 Trp Tyr LysPro Ala Ala Gly Pro His His Tyr Ser Val Gly Arg Ala 5 10 15 Ser Gly LeuLeu Ser Ser Phe His Arg Phe Pro Ser Thr 20 25 6 29 PRT Rattus sp. 6 TrpTyr Lys Pro Ala Ala Gly Ser His His Tyr Ser Val Gly Arg Ala 5 10 15 AlaGly Leu Leu Ser Ser Phe His Arg Phe Pro Ser Thr 20 25 7 13 PRT Homosapiens 7 Trp Tyr Lys Pro Ala Ala Gly His Ser Ser Tyr Ser Val 5 10 8 14PRT Homo sapiens 8 Trp Tyr Lys Pro Ala Ala Gly His Ser Ser Tyr Ser ValGly 5 10 9 13 PRT Mus sp. 9 Trp Tyr Lys Pro Ala Ala Gly Pro His His TyrSer Val 5 10 10 14 PRT Mus sp. 10 Trp Tyr Lys Pro Ala Ala Gly Pro HisHis Tyr Ser Val Gly 5 10 11 13 PRT Rattus sp. 11 Trp Tyr Lys Pro Ala AlaGly Ser His His Tyr Ser Val 5 10 12 14 PRT Rattus sp. 12 Trp Tyr Lys ProAla Ala Gly Ser His His Tyr Ser Val Gly 5 10 13 70 PRT Homo sapiens 13Ser Gln Pro Tyr Arg Gly Ala Glu Pro Pro Gly Gly Ala Gly Ala Ser 5 10 15Pro Glu Leu Gln Leu His Pro Arg Leu Arg Ser Leu Ala Val Cys Val 20 25 30Gln Asp Val Ala Pro Asn Leu Gln Arg Cys Glu Arg Leu Pro Asp Gly 35 40 45Arg Gly Thr Tyr Gln Cys Lys Ala Asn Val Phe Leu Ser Leu Arg Ala 50 55 60Ala Asp Cys Leu Ala Ala 65 70 14 67 PRT Mus sp. 14 Ser Glu Ser Pro AlaLeu Arg Val Gly Thr Gly Pro Leu Arg Asn Leu 5 10 15 Glu Met Arg Pro SerVal Arg Ser Leu Ala Leu Cys Val Lys Asp Val 20 25 30 Thr Pro Asn Leu GlnSer Cys Gln Arg Gln Leu Asn Ser Arg Gly Thr 35 40 45 Phe Gln Cys Lys AlaAsp Val Phe Leu Ser Leu His Glu Thr Asp Cys 50 55 60 Gln Ser Thr 65 1567 PRT Rattus sp. 15 Ser Glu Ser Pro Ala Leu Arg Val Gly Thr Val Pro LeuArg Asn Leu 5 10 15 Glu Met Arg Pro Ser Val Arg Ser Leu Ala Leu Cys ValLys Asp Val 20 25 30 Thr Pro Asn Leu Gln Ser Cys Gln Arg Gln Leu Asn SerArg Gly Thr 35 40 45 Phe Gln Cys Lys Ala Asp Val Phe Leu Ser Leu His LysAla Glu Cys 50 55 60 Gln Ser Ala 65 16 44 PRT Homo sapiens 16 Ser LeuAla Val Cys Val Gln Asp Val Ala Pro Asn Leu Gln Arg Cys 5 10 15 Glu ArgLeu Pro Asp Gly Arg Gly Thr Tyr Gln Cys Lys Ala Asn Val 20 25 30 Phe LeuSer Leu Arg Ala Ala Asp Cys Leu Ala Ala 35 40 17 44 PRT Mus sp. 17 SerLeu Ala Leu Cys Val Lys Asp Val Thr Pro Asn Leu Gln Ser Cys 5 10 15 GlnArg Gln Leu Asn Ser Arg Gly Thr Phe Gln Cys Lys Ala Asp Val 20 25 30 PheLeu Ser Leu His Glu Thr Asp Cys Gln Ser Thr 35 40 18 44 PRT Rattus sp.18 Ser Leu Ala Leu Cys Val Lys Asp Val Thr Pro Asn Leu Gln Ser Cys 5 1015 Gln Arg Gln Leu Asn Ser Arg Gly Thr Phe Gln Cys Lys Ala Asp Val 20 2530 Phe Leu Ser Leu His Lys Ala Glu Cys Gln Ser Ala 35 40 19 101 PRT Homosapiens 19 Trp Tyr Lys Pro Ala Ala Gly His Ser Ser Tyr Ser Val Gly ArgAla 5 10 15 Ala Gly Leu Leu Ser Gly Leu Arg Arg Ser Pro Tyr Ala Arg ArgSer 20 25 30 Gln Pro Tyr Arg Gly Ala Glu Pro Pro Gly Gly Ala Gly Ala SerPro 35 40 45 Glu Leu Gln Leu His Pro Arg Leu Arg Ser Leu Ala Val Cys ValGln 50 55 60 Asp Val Ala Pro Asn Leu Gln Arg Cys Glu Arg Leu Pro Asp GlyArg 65 70 75 80 Gly Thr Tyr Gln Cys Lys Ala Asn Val Phe Leu Ser Leu ArgAla Ala 85 90 95 Asp Cys Leu Ala Ala 100 20 98 PRT Mus sp. 20 Trp TyrLys Pro Ala Ala Gly Pro His His Tyr Ser Val Gly Arg Ala 5 10 15 Ser GlyLeu Leu Ser Ser Phe His Arg Phe Pro Ser Thr Arg Arg Ser 20 25 30 Glu SerPro Ala Leu Arg Val Gly Thr Gly Pro Leu Arg Asn Leu Glu 35 40 45 Met ArgPro Ser Val Arg Ser Leu Ala Leu Cys Val Lys Asp Val Thr 50 55 60 Pro AsnLeu Gln Ser Cys Gln Arg Gln Leu Asn Ser Arg Gly Thr Phe 65 70 75 80 GlnCys Lys Ala Asp Val Phe Leu Ser Leu His Glu Thr Asp Cys Gln 85 90 95 SerThr 21 98 PRT Rattus sp. 21 Trp Tyr Lys Pro Ala Ala Gly Ser His His TyrSer Val Gly Arg Ala 5 10 15 Ala Gly Leu Leu Ser Ser Phe His Arg Phe ProSer Thr Arg Arg Ser 20 25 30 Glu Ser Pro Ala Leu Arg Val Gly Thr Val ProLeu Arg Asn Leu Glu 35 40 45 Met Arg Pro Ser Val Arg Ser Leu Ala Leu CysVal Lys Asp Val Thr 50 55 60 Pro Asn Leu Gln Ser Cys Gln Arg Gln Leu AsnSer Arg Gly Thr Phe 65 70 75 80 Gln Cys Lys Ala Asp Val Phe Leu Ser LeuHis Lys Ala Glu Cys Gln 85 90 95 Ser Ala 22 125 PRT Homo sapiens 22 MetAla Arg Ser Ala Thr Leu Ala Ala Ala Ala Leu Ala Leu Cys Leu 5 10 15 LeuLeu Ala Pro Pro Gly Leu Ala Trp Tyr Lys Pro Ala Ala Gly His 20 25 30 SerSer Tyr Ser Val Gly Arg Ala Ala Gly Leu Leu Ser Gly Leu Arg 35 40 45 ArgSer Pro Tyr Ala Arg Arg Ser Gln Pro Tyr Arg Gly Ala Glu Pro 50 55 60 ProGly Gly Ala Gly Ala Ser Pro Glu Leu Gln Leu His Pro Arg Leu 65 70 75 80Arg Ser Leu Ala Val Cys Val Gln Asp Val Ala Pro Asn Leu Gln Arg 85 90 95Cys Glu Arg Leu Pro Asp Gly Arg Gly Thr Tyr Gln Cys Lys Ala Asn 100 105110 Val Phe Leu Ser Leu Arg Ala Ala Asp Cys Leu Ala Ala 115 120 125 23119 PRT Mus sp. 23 Met Ala Arg Cys Arg Thr Leu Val Ala Ala Ala Leu AlaLeu Leu Leu 5 10 15 Pro Pro Ala Leu Ala Trp Tyr Lys Pro Ala Ala Gly ProHis His Tyr 20 25 30 Ser Val Gly Arg Ala Ser Gly Leu Leu Ser Ser Phe HisArg Phe Pro 35 40 45 Ser Thr Arg Arg Ser Glu Ser Pro Ala Leu Arg Val GlyThr Gly Pro 50 55 60 Leu Arg Asn Leu Glu Met Arg Pro Ser Val Arg Ser LeuAla Leu Cys 65 70 75 80 Val Lys Asp Val Thr Pro Asn Leu Gln Ser Cys GlnArg Gln Leu Asn 85 90 95 Ser Arg Gly Thr Phe Gln Cys Lys Ala Asp Val PheLeu Ser Leu His 100 105 110 Glu Thr Asp Cys Gln Ser Thr 115 24 119 PRTRattus sp. 24 Met Val Arg Cys Arg Thr Leu Val Ala Ala Ala Leu Ala LeuLeu Leu 5 10 15 Thr Pro Ala Leu Ala Trp Tyr Lys Pro Ala Ala Gly Ser HisHis Tyr 20 25 30 Ser Val Gly Arg Ala Ala Gly Leu Leu Ser Ser Phe His ArgPhe Pro 35 40 45 Ser Thr Arg Arg Ser Glu Ser Pro Ala Leu Arg Val Gly ThrVal Pro 50 55 60 Leu Arg Asn Leu Glu Met Arg Pro Ser Val Arg Ser Leu AlaLeu Cys 65 70 75 80 Val Lys Asp Val Thr Pro Asn Leu Gln Ser Cys Gln ArgGln Leu Asn 85 90 95 Ser Arg Gly Thr Phe Gln Cys Lys Ala Asp Val Phe LeuSer Leu His 100 105 110 Lys Ala Glu Cys Gln Ser Ala 115 25 69 DNA Homosapiens 25 tggtacaagc cagcggcggg gcacagctcc tactcggtgg gccgcgccgcggggctgctg 60 tccggcctc 69 26 72 DNA Mus sp. 26 tggtacaagc ccgcggcgggaccccaccac tactcggtgg gccgcgcctc ggggctactg 60 tcgagtttcc ac 72 27 72DNA Rattus sp. 27 tggtacaagc ccgcggcggg atcccaccac tactcggtgg gccgcgctgcggggctactg 60 tcgagtttcc ac 72 28 87 DNA Homo sapiens 28 tggtacaagccagcggcggg gcacagctcc tactcggtgg gccgcgccgc ggggctgctg 60 tccggcctccgcaggtcccc gtacgcg 87 29 87 DNA Mus sp. 29 tggtacaagc ccgcggcgggaccccaccac tactcggtgg gccgcgcctc ggggctactg 60 tcgagtttcc acaggttcccgtccacg 87 30 87 DNA Rattus sp. 30 tggtacaagc ccgcggcggg atcccaccactactcggtgg gccgcgctgc ggggctactg 60 tcgagtttcc acaggttccc atccacg 87 3139 DNA Homo sapiens 31 tggtacaagc cagcggcggg gcacagctcc tactcggtg 39 3242 DNA Homo sapiens 32 tggtacaagc cagcggcggg gcacagctcc tactcggtgg gc 4233 39 DNA Mus sp. 33 tggtacaagc ccgcggcggg accccaccac tactcggtg 39 34 42DNA Mus sp. 34 tggtacaagc ccgcggcggg accccaccac tactcggtgg gc 42 35 39DNA Rattus sp. 35 tggtacaagc ccgcggcggg atcccaccac tactcggtg 39 36 42DNA Rattus sp. 36 tggtacaagc ccgcggcggg atcccaccac tactcggtgg gc 42 37210 DNA Homo sapiens 37 tcccagccct acagaggggc ggaacccccg ggcggggccggcgcctcccc ggagctgcaa 60 ctgcacccca ggctgcggag cctcgctgtg tgcgtccaggacgtcgcccc aaacctgcag 120 aggtgcgagc ggctccccga cggccgcggg acctaccagtgcaaggcgaa cgtcttcctg 180 tccctgcgcg cagccgactg cctcgccgcc 210 38 201DNA Mus sp. 38 tccgagtctc cagcactccg ggtgggaacc ggacctctgc gcaatttagagatgcgcccc 60 agcgtaagga gccttgccct gtgtgtcaaa gatgtgaccc cgaacctgcagagctgccag 120 cggcaactca acagccgagg gactttccag tgtaaagcgg acgtcttcttgtcgctgcac 180 gagactgatt gccagagcac c 201 39 201 DNA Rattus sp. 39tccgagtctc cagcactccg ggtgggaacc gtacctctgc gcaacttgga gatgcgccca 60agcgtaagaa gccttgccct gtgtgtcaaa gatgtgaccc cgaacctgca gagctgccag 120cggcaactca acagccgagg gactttccag tgtaaggcgg acgtcttctt gtcgctgcac 180aaggctgaat gccaaagcgc c 201 40 132 DNA Homo sapiens 40 agcctcgctgtgtgcgtcca ggacgtcgcc ccaaacctgc agaggtgcga gcggctcccc 60 gacggccgcgggacctacca gtgcaaggcg aacgtcttcc tgtccctgcg cgcagccgac 120 tgcctcgccg cc132 41 132 DNA Mus sp. 41 agccttgccc tgtgtgtcaa agatgtgacc ccgaacctgcagagctgcca gcggcaactc 60 aacagccgag ggactttcca gtgtaaagcg gacgtcttcttgtcgctgca cgagactgat 120 tgccagagca cc 132 42 132 DNA Rattus sp. 42agccttgccc tgtgtgtcaa agatgtgacc ccgaacctgc agagctgcca gcggcaactc 60aacagccgag ggactttcca gtgtaaggcg gacgtcttct tgtcgctgca caaggctgaa 120tgccaaagcg cc 132 43 303 DNA Homo sapiens 43 tggtacaagc cagcggcggggcacagctcc tactcggtgg gccgcgccgc ggggctgctg 60 tccggcctcc gcaggtccccgtacgcgcgg cgctcccagc cctacagagg ggcggaaccc 120 ccgggcgggg ccggcgcctccccggagctg caactgcacc ccaggctgcg gagcctcgct 180 gtgtgcgtcc aggacgtcgccccaaacctg cagaggtgcg agcggctccc cgacggccgc 240 gggacctacc agtgcaaggcgaacgtcttc ctgtccctgc gcgcagccga ctgcctcgcc 300 gcc 303 44 294 DNA Mussp. 44 tggtacaagc ccgcggcggg accccaccac tactcggtgg gccgcgcctc ggggctactg60 tcgagtttcc acaggttccc gtccacgcga cgctccgagt ctccagcact ccgggtggga 120accggacctc tgcgcaattt agagatgcgc cccagcgtaa ggagccttgc cctgtgtgtc 180aaagatgtga ccccgaacct gcagagctgc cagcggcaac tcaacagccg agggactttc 240cagtgtaaag cggacgtctt cttgtcgctg cacgagactg attgccagag cacc 294 45 294DNA Rattus sp. 45 tggtacaagc ccgcggcggg atcccaccac tactcggtgg gccgcgctgcggggctactg 60 tcgagtttcc acaggttccc atccacgcga cgttccgagt ctccagcactccgggtggga 120 accgtacctc tgcgcaactt ggagatgcgc ccaagcgtaa gaagccttgccctgtgtgtc 180 aaagatgtga ccccgaacct gcagagctgc cagcggcaac tcaacagccgagggactttc 240 cagtgtaagg cggacgtctt cttgtcgctg cacaaggctg aatgccaaagcgcc 294 46 375 DNA Homo sapiens 46 atggcccggt ccgcgacact ggcggccgccgccctggcgc tgtgcctgct gctggcgccg 60 cctggcctcg cgtggtacaa gccagcggcggggcacagct cctactcggt gggccgcgcc 120 gcggggctgc tgtccggcct ccgcaggtccccgtacgcgc ggcgctccca gccctacaga 180 ggggcggaac ccccgggcgg ggccggcgcctccccggagc tgcaactgca ccccaggctg 240 cggagcctcg ctgtgtgcgt ccaggacgtcgccccaaacc tgcagaggtg cgagcggctc 300 cccgacggcc gcgggaccta ccagtgcaaggcgaacgtct tcctgtccct gcgcgcagcc 360 gactgcctcg ccgcc 375 47 357 DNA Mussp. 47 atggcccggt gtaggacgct ggtggccgct gccctggcgc tgctcctgcc gccagccctc60 gcgtggtaca agcccgcggc gggaccccac cactactcgg tgggccgcgc ctcggggcta 120ctgtcgagtt tccacaggtt cccgtccacg cgacgctccg agtctccagc actccgggtg 180ggaaccggac ctctgcgcaa tttagagatg cgccccagcg taaggagcct tgccctgtgt 240gtcaaagatg tgaccccgaa cctgcagagc tgccagcggc aactcaacag ccgagggact 300ttccagtgta aagcggacgt cttcttgtcg ctgcacgaga ctgattgcca gagcacc 357 48357 DNA Rattus sp. 48 atggtccggt gtaggacgct ggtggccgcc gccctggcgctgctcctgac gccagccctc 60 gcgtggtaca agcccgcggc gggatcccac cactactcggtgggccgcgc tgcggggcta 120 ctgtcgagtt tccacaggtt cccatccacg cgacgttccgagtctccagc actccgggtg 180 ggaaccgtac ctctgcgcaa cttggagatg cgcccaagcgtaagaagcct tgccctgtgt 240 gtcaaagatg tgaccccgaa cctgcagagc tgccagcggcaactcaacag ccgagggact 300 ttccagtgta aggcggacgt cttcttgtcg ctgcacaaggctgaatgcca aagcgcc 357 49 328 PRT Homo sapiens 49 Met Asp Asn Ala SerPhe Ser Glu Pro Trp Pro Ala Asn Ala Ser Gly 1 5 10 15 Pro Asp Pro AlaLeu Ser Cys Ser Asn Ala Ser Thr Leu Ala Pro Leu 20 25 30 Pro Ala Pro LeuAla Val Ala Val Pro Val Val Tyr Ala Val Ile Cys 35 40 45 Ala Val Gly LeuAla Gly Asn Ser Ala Val Leu Tyr Val Leu Leu Arg 50 55 60 Ala Pro Arg MetLys Thr Val Thr Asn Leu Phe Ile Leu Asn Leu Ala 65 70 75 80 Ile Ala AspGlu Leu Phe Thr Leu Val Leu Pro Ile Asn Ile Ala Asp 85 90 95 Phe Leu LeuArg Gln Trp Pro Phe Gly Glu Leu Met Cys Lys Leu Ile 100 105 110 Val AlaIle Asp Gln Tyr Asn Thr Phe Ser Ser Leu Tyr Phe Leu Thr 115 120 125 ValMet Ser Ala Asp Arg Tyr Leu Val Val Leu Ala Thr Ala Glu Ser 130 135 140Arg Arg Val Ala Gly Arg Thr Tyr Ser Ala Ala Arg Ala Val Ser Leu 145 150155 160 Ala Val Trp Gly Ile Val Thr Leu Val Val Leu Pro Phe Ala Val Phe165 170 175 Ala Arg Leu Asp Asp Glu Gln Gly Arg Arg Gln Cys Val Leu ValPhe 180 185 190 Pro Gln Pro Glu Ala Phe Trp Trp Arg Ala Ser Arg Leu TyrThr Leu 195 200 205 Val Leu Gly Phe Ala Ile Pro Val Ser Thr Ile Cys ValLeu Tyr Thr 210 215 220 Thr Leu Leu Cys Arg Leu His Ala Met Arg Leu AspSer His Ala Lys 225 230 235 240 Ala Leu Glu Arg Ala Lys Lys Arg Val ThrPhe Leu Val Val Ala Ile 245 250 255 Leu Ala Val Cys Leu Leu Cys Trp ThrPro Tyr His Leu Ser Thr Val 260 265 270 Val Ala Leu Thr Thr Asp Leu ProGln Thr Pro Leu Val Ile Ala Ile 275 280 285 Ser Tyr Phe Ile Thr Ser LeuSer Tyr Ala Asn Ser Cys Leu Asn Pro 290 295 300 Phe Leu Tyr Ala Phe LeuAsp Ala Ser Phe Arg Arg Asn Leu Arg Gln 305 310 315 320 Leu Ile Thr CysArg Ala Ala Ala 325 50 1000 DNA Homo sapiens 50 atcgatatgg acaacgcctcgttctcggag ccctggcccg ccaacgcatc gggcccggac 60 ccggcgctga gctgctccaacgcgtcgact ctggcgccgc tgccggcgcc gctggcggtg 120 gctgtaccag ttgtctacgcggtgatctgc gccgtgggtc tggcgggcaa ctccgccgtg 180 ctgtacgtgt tgctgcgggcgccccgcatg aagaccgtca ccaacctgtt catcctcaac 240 ctggccatcg ccgacgagctcttcacgctg gtgctgccca tcaacatcgc cgacttcctg 300 ctgcggcagt ggcccttcggggagctcatg tgcaagctca tcgtggctat cgaccagtac 360 aacaccttct ccagcctctacttcctcacc gtcatgagcg ccgaccgcta cctggtggtg 420 ttggccactg cggagtcgcgccgggtggcc ggccgcacct acagcgccgc gcgcgcggtg 480 agcctggccg tgtgggggatcgtcacactc gtcgtgctgc ccttcgcagt cttcgcccgg 540 ctagacgacg agcagggccggcgccagtgc gtgctagtct ttccgcagcc cgaggccttc 600 tggtggcgcg cgagccgcctctacacgctc gtgctgggct tcgccatccc cgtgtccacc 660 atctgtgtcc tctataccaccctgctgtgc cggctgcatg ccatgcggct ggacagccac 720 gccaaggccc tggagcgcgccaagaagcgg gtgaccttcc tggtggtggc aatcctggcg 780 gtgtgcctcc tctgctggacgccctaccac ctgagcaccg tggtggcgct caccaccgac 840 ctcccgcaga cgccgctggtcatcgctatc tcctacttca tcaccagcct gagctacgcc 900 aacagctgcc tcaaccccttcctctacgcc ttcctggacg ccagcttccg caggaacctc 960 cgccagctga taacttgccgcgcggcagcc tgacactagt 1000 51 33 DNA Artificial Sequence Primer 51gtcgacatgg cccggtccgc gacactggcg gcc 33 52 33 DNA Artificial SequencePrimer 52 gctagcagcg gtgccaggag aggtccgggc tca 33 53 33 DNA ArtificialSequence Primer 53 gtcgacagct ccatggcccg gtgtaggacg ctg 33 54 33 DNAArtificial Sequence Primer 54 gctagctcag gtgctctggc aatcagtctc gtg 33 5527 DNA Artificial Sequence Primer 55 cacggctcca tggtccggtg taggacg 27 5627 DNA Artificial Sequence Primer 56 cagcgtcgag gtttgggttg gggttca 27 5732 DNA Artificial Sequence Primer 57 atcgatatgg acaacgcctc gttctcggag cc32 58 32 DNA Artificial Sequence Primer 58 actagtgtca ggctgccgcgcggcaagtta tc 32 59 329 PRT Rattus sp. 59 Met His Asn Leu Ser Leu PheGlu Pro Gly Arg Gly Asn Val Ser Cys 5 10 15 Gly Gly Pro Phe Leu Gly CysPro Asn Glu Ser Asn Pro Ala Pro Leu 20 25 30 Pro Leu Pro Gln Pro Leu AlaVal Ala Val Pro Val Val Tyr Gly Val 35 40 45 Ile Cys Ala Val Gly Leu AlaGly Asn Ser Ala Val Leu Tyr Val Leu 50 55 60 Leu Arg Thr Pro Arg Met LysThr Val Thr Asn Val Phe Ile Leu Asn 65 70 75 80 Leu Ala Ile Ala Asp GluLeu Phe Thr Leu Val Leu Pro Ile Asn Ile 85 90 95 Ala Asp Phe Leu Leu ArgArg Trp Pro Phe Gly Glu Val Met Cys Lys 100 105 110 Leu Ile Val Ala ValAsp Gln Tyr Asn Thr Phe Ser Ser Leu Tyr Phe 115 120 125 Leu Ala Val MetSer Ala Asp Arg Tyr Leu Val Val Leu Ala Thr Ala 130 135 140 Glu Ser ArgArg Val Ser Gly Arg Thr Tyr Gly Ala Ala Arg Ala Val 145 150 155 160 SerLeu Ala Val Trp Ala Leu Val Thr Leu Val Val Leu Pro Phe Ala 165 170 175Val Phe Ala Arg Leu Asp Glu Glu Gln Gly Arg Arg Gln Cys Val Leu 180 185190 Val Phe Pro Gln Pro Glu Ala Phe Trp Trp Arg Ala Ser Arg Leu Tyr 195200 205 Thr Leu Val Leu Gly Phe Ala Ile Pro Val Ser Thr Ile Cys Ala Leu210 215 220 Tyr Ile Thr Leu Leu Cys Arg Leu Arg Ala Ile Gln Leu Asp SerHis 225 230 235 240 Ala Lys Ala Leu Asp Arg Ala Lys Lys Arg Val Thr LeuLeu Val Val 245 250 255 Ala Ile Leu Ala Val Cys Leu Leu Cys Trp Thr ProTyr His Leu Ser 260 265 270 Thr Ile Val Ala Leu Thr Thr Asp Leu Pro GlnThr Pro Leu Val Ile 275 280 285 Gly Ile Ser Tyr Phe Ile Thr Ser Leu SerTyr Ala Asn Ser Cys Leu 290 295 300 Asn Pro Phe Leu Tyr Ala Phe Leu AspAsp Ser Phe Arg Arg Ser Leu 305 310 315 320 Arg Gln Leu Val Ser Cys ArgThr Ala 325 60 987 DNA Rattus sp. 60 atgcacaact tgtcgctctt cgagcctggcaggggcaatg tgtcttgcgg cggcccattt 60 ttgggctgtc ctaacgagtc gaacccagcgcctctgccac tgccgcagcc tctggcggta 120 gcagtgcctg tggtctacgg ggtgatctgcgcggtgggac tggcgggcaa ctccgcggtg 180 ctgtacgtac tgctgcgcac gccgcgcatgaagactgtta ccaacgtgtt cattctcaac 240 ctggctatcg cggacgagct cttcaccctcgtgctgccca tcaacatcgc ggacttcctg 300 ctgaggcgct ggcccttcgg ggaagtcatgtgcaagctca tcgtggctgt cgaccagtac 360 aacactttct ctagcctcta cttcctcgccgtcatgagcg cagaccgcta cctggttgtc 420 ctggccacag ccgagtcgcg ccgggtgtccgggcgcactt atggtgcagc gcgggctgtc 480 agtctggcgg tgtgggcgct ggtgacattggtcgtgctgc cttttgcggt attcgcccgg 540 ctggacgaag agcagggtcg gcgtcagtgcgtgctggtct tcccgcagcc tgaggccttc 600 tggtggcgcg ccagccgtct gtacactctagtgttgggct tcgccatccc ggtgtccacc 660 atctgcgccc tctatatcac cctgttgtgccgactgcgtg ctatccagct agacagccac 720 gccaaggccc tggaccgtgc caagaagcgcgtgaccttgt tggtggtggc gattctggct 780 gtgtgcctcc tctgctggac accgtaccacctgagcacca tagtggcgct caccaccgac 840 ctcccgcaaa caccgttggt catcggcatctcttacttca tcaccagtct gagctatgcc 900 aacagctgcc tcaacccttt cctctatgccttcctggacg acagcttccg caggagcctg 960 cggcagctgg tgtcatgccg cacagcc 98761 28 DNA Artificial Sequence Primer 61 actgatatgc acaacttgtc gctcttcg28 62 28 DNA Artificial Sequence Primer 62 actagttcag gctgtgcggcatgacacc 28 63 22 DNA Artificial Sequence Primer 63 ctgtcgagtttccacaggtt cc 22 64 20 DNA Artificial Sequence Primer 64 ttgcgcagaggtacggttcc 20 65 26 DNA Artificial Sequence Primer 65 cgtgccaagaaacgcgtgac cttgtt 26 66 23 PRT Bos sp. 66 Trp Tyr Lys Pro Thr Ala GlyGln Gly Tyr Tyr Ser Val Gly Arg Ala 1 5 10 15 Ala Gly Leu Leu Ser GlyPhe 20 67 29 PRT Bos sp. 67 Trp Tyr Lys Pro Thr Ala Gly Gln Gly Tyr TyrSer Val Gly Arg Ala 1 5 10 15 Ala Gly Leu Leu Ser Gly Phe His Arg SerPro Tyr Ala 20 25 68 13 PRT Bos sp. 68 Trp Tyr Lys Pro Thr Ala Gly GlnGly Tyr Tyr Ser Val 1 5 10 69 14 PRT Bos sp. 69 Trp Tyr Lys Pro Thr AlaGly Gln Gly Tyr Tyr Ser Val Gly 1 5 10 70 67 PRT Bos sp. 70 Ser Glu ProArg Gly Gly Thr Arg Ser Leu Gly Gly Val Gly Thr Phe 1 5 10 15 Arg GluMet Arg Pro Asn Leu Arg Ser Leu Ala Val Cys Val Glu Glu 20 25 30 Val ThrPro Asn Leu Gln Ser Cys Glu Pro Leu Pro Asp Gly Arg Ala 35 40 45 Thr PheGln Cys Lys Ala Asp Val Phe Leu Ser Leu Ser Ala Ser Asp 50 55 60 Cys ArgLys 65 71 43 PRT Bos sp. 71 Ser Leu Ala Val Cys Val Glu Glu Val Thr ProAsn Leu Gln Ser Cys 1 5 10 15 Glu Pro Leu Pro Asp Gly Arg Ala Thr PheGln Cys Lys Ala Asp Val 20 25 30 Phe Leu Ser Leu Ser Ala Ser Asp Cys ArgLys 35 40 72 98 PRT Bos sp. 72 Trp Tyr Lys Pro Thr Ala Gly Gln Gly TyrTyr Ser Val Gly Arg Ala 1 5 10 15 Ala Gly Leu Leu Ser Gly Phe His ArgSer Pro Tyr Ala Arg Arg Ser 20 25 30 Glu Pro Arg Gly Gly Thr Arg Ser LeuGly Gly Val Gly Thr Phe Arg 35 40 45 Glu Met Arg Pro Asn Leu Arg Ser LeuAla Val Cys Val Glu Glu Val 50 55 60 Thr Pro Asn Leu Gln Ser Cys Glu ProLeu Pro Asp Gly Arg Ala Thr 65 70 75 80 Phe Gln Cys Lys Ala Asp Val PheLeu Ser Leu Ser Ala Ser Asp Cys 85 90 95 Arg Lys 73 122 PRT Bos sp. 73Met Ala Gly Pro Ala Met Leu Val Ala Ala Ala Leu Ala Leu Cys Leu 5 10 15Leu Leu Ala Ser Pro Gly Leu Ala Trp Tyr Lys Pro Thr Ala Gly Gln 20 25 30Gly Tyr Tyr Ser Val Gly Arg Ala Ala Gly Leu Leu Ser Gly Phe His 35 40 45Arg Ser Pro Tyr Ala Arg Arg Ser Glu Pro Arg Gly Gly Thr Arg Ser 50 55 60Leu Gly Gly Val Gly Thr Phe Arg Glu Met Arg Pro Asn Leu Arg Ser 65 70 7580 Leu Ala Val Cys Val Glu Glu Val Thr Pro Asn Leu Gln Ser Cys Glu 85 9095 Pro Leu Pro Asp Gly Arg Ala Thr Phe Gln Cys Lys Ala Asp Val Phe 100105 110 Leu Ser Leu Ser Ala Ser Asp Cys Arg Lys 115 120 74 69 DNA Bossp. 74 tggtacaagc cgacggcggg gcaggggtac tactccgtgg gccgcgccgc ggggctgctg60 tccggcttc 69 75 87 DNA Bos sp. 75 tggtacaagc cgacggcggg gcaggggtactactccgtgg gccgcgccgc ggggctgctg 60 tccggcttcc acaggtcgcc gtacgca 87 7639 DNA Bos sp. 76 tggtacaagc cgacggcggg gcaggggtac tactccgtg 39 77 42DNA Bos sp. 77 tggtacaagc cgacggcggg gcaggggtac tactccgtgg gc 42 78 201DNA Bos sp. 78 tccgagcccc gcgggggcac gcgatccctg ggaggggtcg gcactttccgggagatgcgc 60 cccaacctgc ggagtcttgc cgtgtgcgtc gaggaggtca cccccaacctgcagagctgc 120 gagccactcc ccgacggccg cgccactttc cagtgcaagg ccgacgtcttcctgtcgctc 180 agcgcctcgg actgtcgcaa g 201 79 129 DNA Bos sp. 79agtcttgccg tgtgcgtcga ggaggtcacc cccaacctgc agagctgcga gccactcccc 60gacggccgcg ccactttcca gtgcaaggcc gacgtcttcc tgtcgctcag cgcctcggac 120tgtcgcaag 129 80 294 DNA Bos sp. 80 tggtacaagc cgacggcggg gcaggggtactactccgtgg gccgcgccgc ggggctgctg 60 tccggcttcc acaggtcgcc gtacgcacggcgctccgagc cccgcggggg cacgcgatcc 120 ctgggagggg tcggcacttt ccgggagatgcgccccaacc tgcggagtct tgccgtgtgc 180 gtcgaggagg tcacccccaa cctgcagagctgcgagccac tccccgacgg ccgcgccact 240 ttccagtgca aggccgacgt cttcctgtcgctcagcgcct cggactgtcg caag 294 81 366 DNA Bos sp. 81 atggccgggcccgcgatgct ggtggccgcc gctctggcgc tgtgcttact gctggcgtcc 60 cctggcctcgcgtggtacaa gccgacggcg gggcaggggt actactccgt gggccgcgcc 120 gcggggctgctgtccggctt ccacaggtcg ccgtacgcac ggcgctccga gccccgcggg 180 ggcacgcgatccctgggagg ggtcggcact ttccgggaga tgcgccccaa cctgcggagt 240 cttgccgtgtgcgtcgagga ggtcaccccc aacctgcaga gctgcgagcc actccccgac 300 ggccgcgccactttccagtg caaggccgac gtcttcctgt cgctcagcgc ctcggactgt 360 cgcaag 366 8230 DNA Artificial Sequence Primer 82 cccatggccg ggcccgcgat gctggtcgcc 3083 30 DNA Artificial Sequence Primer 83 tcacttgcga cagtccgagg cgctgagcga30 84 333 PRT Homo sapiens 84 Met Gln Ala Ala Gly His Pro Glu Pro LeuAsp Ser Arg Gly Ser Phe 1 5 10 15 Ser Leu Pro Thr Met Gly Ala Asn ValSer Gln Asp Asn Gly Thr Gly 20 25 30 His Asn Ala Thr Phe Ser Glu Pro LeuPro Phe Leu Tyr Val Leu Leu 35 40 45 Pro Ala Val Tyr Ser Gly Ile Cys AlaVal Gly Leu Thr Gly Asn Thr 50 55 60 Ala Val Ile Leu Val Ile Leu Arg AlaPro Lys Met Lys Thr Val Thr 65 70 75 80 Asn Val Phe Ile Leu Asn Leu AlaVal Ala Asp Gly Leu Phe Thr Leu 85 90 95 Val Leu Pro Val Asn Ile Ala GluHis Leu Leu Gln Tyr Trp Pro Phe 100 105 110 Gly Glu Leu Leu Cys Lys LeuVal Leu Ala Val Asp His Tyr Asn Ile 115 120 125 Phe Ser Ser Ile Tyr PheLeu Ala Val Met Ser Val Asp Arg Tyr Leu 130 135 140 Val Val Leu Ala ThrVal Arg Ser Arg His Met Pro Trp Arg Thr Tyr 145 150 155 160 Arg Gly AlaLys Val Ala Ser Leu Cys Val Trp Leu Gly Val Thr Val 165 170 175 Leu ValLeu Pro Phe Phe Ser Phe Ala Gly Val Tyr Ser Asn Glu Leu 180 185 190 GlnVal Pro Ser Cys Gly Leu Ser Phe Pro Trp Pro Glu Gln Val Trp 195 200 205Phe Lys Ala Ser Arg Val Tyr Thr Leu Val Leu Gly Phe Val Leu Pro 210 215220 Val Cys Thr Ile Cys Val Leu Tyr Thr Asp Leu Leu Arg Arg Leu Arg 225230 235 240 Ala Val Arg Leu Arg Ser Gly Ala Lys Ala Leu Gly Lys Ala ArgArg 245 250 255 Lys Val Thr Val Leu Val Leu Val Val Leu Ala Val Cys LeuLeu Cys 260 265 270 Trp Thr Pro Phe His Leu Ala Ser Val Val Ala Leu ThrThr Asp Leu 275 280 285 Pro Gln Thr Pro Leu Val Ile Ser Met Ser Tyr ValIle Thr Ser Leu 290 295 300 Ser Tyr Ala Asn Ser Cys Leu Asn Pro Phe LeuTyr Ala Phe Leu Asp 305 310 315 320 Asp Asn Phe Arg Lys Asn Phe Arg SerIle Leu Arg Cys 325 330 85 1023 DNA Homo sapiens 85 atcgattacaatgcaggccg ctgggcaccc agagcccctt gacagcaggg gctccttctc 60 cctccccacgatgggtgcca acgtctctca ggacaatggc actggccaca atgccacctt 120 ctccgagccactgccgttcc tctatgtgct cctgcccgcc gtgtactccg ggatctgtgc 180 tgtggggctgactggcaaca cggccgtcat ccttgtaatc ctaagggcgc ccaagatgaa 240 gacggtgaccaacgtgttca tcctgaacct ggccgtcgcc gacgggctct tcacgctggt 300 actgcccgtcaacatcgcgg agcacctgct gcagtactgg cccttcgggg agctgctctg 360 caagctggtgctggccgtcg accactacaa catcttctcc agcatctact tcctagccgt 420 gatgagcgtggaccgatacc tggtggtgct ggccaccgtg aggtcccgcc acatgccctg 480 gcgcacctaccggggggcga aggtcgccag cctgtgtgtc tggctgggcg tcacggtcct 540 ggttctgcccttcttctctt tcgctggcgt ctacagcaac gagctgcagg tcccaagctg 600 tgggctgagcttcccgtggc ccgagcaggt ctggttcaag gccagccgtg tctacacgtt 660 ggtcctgggcttcgtgctgc ccgtgtgcac catctgtgtg ctctacacag acctcctgcg 720 caggctgcgggccgtgcggc tccgctctgg agccaaggct ctaggcaagg ccaggcggaa 780 ggtgaccgtcctggtcctcg tcgtgctggc cgtgtgcctc ctctgctgga cgcccttcca 840 cctggcctctgtcgtggccc tgaccacgga cctgccccag accccactgg tcatcagtat 900 gtcctacgtcatcaccagcc tcagctacgc caactcgtgc ctgaacccct tcctctacgc 960 ctttctagatgacaacttcc ggaagaactt ccgcagcata ttgcggtgct gaagggcact 1020 agt 1023 86331 PRT Bos sp. 86 Met His Asn Ala Ser Tyr Trp Gly Pro Glu Arg Ala AsnThr Ser Cys 5 10 15 Pro Ala Pro Ala Pro Thr Leu Gly Cys Pro Asn Ala SerGly Pro Ala 20 25 30 Pro Pro Leu Pro Pro Pro Leu Ala Val Ala Val Pro ValVal Tyr Ala 35 40 45 Val Ile Cys Ala Val Gly Leu Ala Gly Asn Ser Ala ValLeu Phe Val 50 55 60 Leu Leu Arg Ala Pro Arg Arg Lys Thr Val Thr Asn LeuPhe Ile Leu 65 70 75 80 Asn Leu Ala Val Ala Asp Glu Leu Phe Thr Leu ValPro Pro Val Asn 85 90 95 Ile Ala Asp Phe Leu Leu Arg Arg Trp Pro Phe GlyGlu Leu Leu Cys 100 105 110 Lys Leu Val Val Ala Val Asp Gln Tyr Asn ThrPhe Ser Ser Leu Tyr 115 120 125 Phe Leu Thr Val Met Ser Ala Asp Arg TyrLeu Val Val Leu Ala Thr 130 135 140 Ala Glu Ser Arg Arg Val Ala Gly ArgThr Tyr Gly Ala Ala Arg Ala 145 150 155 160 Val Ser Leu Ala Val Trp GlyVal Ala Thr Leu Val Val Leu Pro Phe 165 170 175 Ala Val Phe Ala Arg LeuAsp Glu Glu Gln Gly Arg Arg Gln Cys Val 180 185 190 Leu Val Phe Pro GlnPro Glu Ala Leu Trp Trp Arg Ala Ser Arg Leu 195 200 205 Tyr Thr Leu ValLeu Gly Phe Ala Ile Pro Val Ser Thr Ile Cys Val 210 215 220 Leu Tyr ThrSer Leu Leu Cys Arg Leu Arg Ala Ile Arg Leu Asp Ser 225 230 235 240 HisAla Lys Ala Leu Asp Arg Ala Lys Lys Arg Val Thr Val Leu Val 245 250 255Val Ala Ile Leu Ala Val Cys Leu Leu Val Trp Thr Pro Tyr His Leu 260 265270 Ser Thr Val Val Ala Leu Thr Thr Asp Leu Pro Gln Thr Pro Leu Val 275280 285 Ile Ala Val Ser Tyr Phe Ile Thr Ser Leu Ser Tyr Ala Asn Ser Cys290 295 300 Leu Asn Pro Phe Leu Tyr Ala Phe Leu Asp Asp Ser Phe Arg ArgSer 305 310 315 320 Leu Arg Gln Leu Leu Ala Cys Arg Thr Thr Ser 325 33087 993 DNA Bos sp. 87 atgcacaacg cgtcgtactg ggggccggag cgcgccaacacgtcgtgccc cgcgcccgca 60 cccacgctcg gctgtcccaa cgcgtccggg ccggcgccgccgctgccgcc gccgctggcc 120 gtagccgtgc ccgttgtgta cgcggtgatc tgcgcagtgggactggcggg caactcggcg 180 gtactgttcg tgctgctgcg ggcgccgcgc aggaagaccgtcaccaacct gttcatcctc 240 aacctggccg tggccgacga gcttttcacg ctcgtgccgcctgtcaacat cgccgacttt 300 ctgctgaggc gctggccctt cggggagctc ctatgcaagctcgtcgtggc cgtcgatcag 360 tacaacacct tctccagcct ctatttcctc acggtcatgagcgccgaccg ctacctggtg 420 gtgctggcca ccgccgagtc gcgccgggtg gccggccgcacgtacggcgc cgcgcgcgcg 480 gtgagcctgg ccgtctgggg ggtcgcgacc ctggtggtgctgcccttcgc ggtgttcgcg 540 cggctcgacg aggagcaggg ccggcgccag tgcgtactggtcttcccgca gcccgaggcc 600 ttgtggtggc gcgcgagccg cctgtacacg ctggtgctcggcttcgccat cccagtgtcc 660 accatctgcg tcctctacac ctcgctgctg tgccggctgcgcgccatacg cctcgacagc 720 cacgccaagg ccctggaccg cgccaagaag cgggtgaccgtcctggtggt ggccatcctg 780 gccgtgtgcc tcctcgtctg gacgccctac cacctgagcaccgtggtggc gctcaccacc 840 gacctcccgc agacgccgct ggtcatcgcc gtgtcctacttcatcaccag cctgagctac 900 gccaacagct gcctcaaccc tttcctctac gccttcctggacgacagctt ccgccggagc 960 ctccgccagc tgctggcgtg ccgcaccacc tcc 993 88336 PRT Bos sp. 88 Met Met Glu Ala Thr Gly Leu Glu Gly Leu Glu Ser ThrSer Ser Pro 5 10 15 Cys Pro Gly Ser Thr Gly Thr Gly Leu Ser Trp Asp AsnGly Thr Arg 20 25 30 His Asn Ala Thr Phe Pro Glu Pro Leu Pro Ala Leu TyrVal Leu Leu 35 40 45 Pro Val Val Tyr Ser Val Ile Cys Ala Val Gly Leu ValGly Asn Ala 50 55 60 Ala Val Ile Cys Val Ile Leu Arg Ala Pro Lys Met LysThr Val Thr 65 70 75 80 His Val Phe Ile Leu Asn Leu Ala Ile Ala Asp GlyLeu Phe Thr Leu 85 90 95 Val Leu Pro Thr Asn Ile Ala Glu His Leu Leu GlnArg Trp Pro Phe 100 105 110 Gly Glu Val Leu Cys Lys Leu Val Leu Ala IleAsp His Cys Asn Ile 115 120 125 Phe Ser Ser Val Tyr Phe Leu Ala Ala MetSer Ile Asp Arg Tyr Leu 130 135 140 Val Val Leu Ala Thr Ala Arg Ser ArgArg Met Pro Arg Arg Thr Val 145 150 155 160 His Arg Ala Lys Val Ala SerLeu Cys Val Trp Leu Gly Val Thr Val 165 170 175 Ala Val Leu Pro Phe LeuThr Phe Ala Gly Val Tyr Asn Asn Glu Leu 180 185 190 Gln Val Thr Ser CysGly Leu Ser Phe Pro Arg Pro Glu Arg Ala Trp 195 200 205 Phe Gln Ala SerArg Ile Tyr Thr Leu Val Leu Gly Phe Val Val Pro 210 215 220 Met Cys ThrLeu Cys Val Leu Tyr Ala Asp Leu Leu Arg Arg Leu Arg 225 230 235 240 AlaLeu Arg Leu His Ser Gly Ala Lys Ala Leu Gly Lys Ala Lys Arg 245 250 255Lys Val Ser Leu Leu Val Leu Ala Val Leu Ala Val Gly Leu Leu Cys 260 265270 Trp Thr Pro Phe His Leu Ala Ser Ile Val Ala Leu Thr Thr Asp Leu 275280 285 Pro Gln Thr Pro Leu Val Ile Ile Val Ser Tyr Val Val Thr Ser Leu290 295 300 Ser Tyr Thr Ser Ser Cys Leu Asn Pro Phe Leu Tyr Ala Phe LeuAsp 305 310 315 320 His Ser Phe Arg Lys Ser Leu Arg Thr Ala Cys Arg CysGln Gly Ala 325 330 335 89 1008 DNA Bos sp. 89 atgatggagg ccactgggctggaaggcctg gaaagcacaa gctccccctg cccaggtagc 60 acaggcaccg gcctctcatgggacaatggc accagacaca atgccacctt ccccgagccg 120 ctgcctgccc tctacgtgctgctgccggtg gtatactctg tcatctgtgc cgtggggctg 180 gtgggcaacg cagccgtcatctgtgtgatc ctgagggctc ccaagatgaa gacagtgacc 240 cacgtgttca tcctgaacctggccatcgcc gacgggctct tcacgctggt gctgcccacc 300 aatattgctg agcacctgctgcagcgctgg ccctttgggg aggtgctctg caagctggtg 360 ctggccattg accactgcaacatcttctcc agtgtctact tcctggccgc catgagtata 420 gaccgctacc tggtggttctggccacggca cgctcccgcc gcatgccccg gcgcaccgtc 480 cacagggcga aggtcgccagcctgtgcgtc tggctgggtg tcacagtcgc agtgctgccc 540 ttccttacct tcgcaggcgtgtacaacaat gagctgcagg tcacaagttg tgggctgagt 600 ttcccgcggc ccgagagggcctggttccag gcaagccgca tctacacgct ggtgctgggc 660 ttcgtggtgc ccatgtgcaccctctgcgtg ctctacgcag acctgctgcg gaggctaagg 720 gccctgcggc tccactccggagccaaggct ctgggcaagg ccaagcggaa ggttagcctc 780 ctggtcctgg ccgtgctggccgtgggcctg ctctgctgga cgcccttcca cctggcctca 840 attgtggccc tgaccacagacctgccccag acaccgctgg tcatcatcgt ctcctatgtg 900 gtcaccagcc tcagctacaccagctcctgc ctcaacccct tcctctatgc cttcctggat 960 cacagcttcc ggaagagcctccgcaccgca tgtcggtgcc agggggca 1008 90 18 DNA Artificial Sequence Primer90 cgctcccagc cctacaga 18 91 20 DNA Artificial Sequence Primer 91tcgccttgca ctggtaggtc 20 92 24 DNA Artificial Sequence Primer 92agcctcgctg tgtgcgtcca ggac 24 93 22 DNA Artificial Sequence Primer 93tgcgtgctat ccagctagac ag 22 94 22 DNA Artificial Sequence Primer 94agaggaggca cacagccaga at 22 95 26 DNA Artificial Sequence Primer 95cgtgccaaga aacgcgtgac cttgtt 26 96 30 DNA Artificial Sequence Primer 96gtcgaccgag tgtctgtcct cgccaggatg 30 97 33 DNA Artificial Sequence Primer97 gctagctcct tgttatcggg ctcaggaggt ggt 33 98 33 DNA Artificial SequencePrimer 98 gtcgaccatg atggaggcca ctgggctgga agg 33 99 33 DNA ArtificialSequence Primer 99 gctagcttat gccccctggc accgacatgc ggt 33 100 23 PRTHomo sapiens 100 Trp Tyr Lys His Val Ala Ser Pro Arg Tyr His Thr Val GlyArg Ala 1 5 10 15 Ala Gly Leu Leu Met Gly Leu 20 101 378 DNA Homosapiens 101 atggcccggt ccgcgacact ggcggccgcc gccctggcgc tgtgcctgctgctggcgccg 60 cctggcctcg cgtggtacaa gccagcggcg gggcacagct cctactcggtgggccgcgcc 120 gcggggctgc tgtccggcct ccgcaggtcc ccgtacgcgc ggcgctcccagccctacaga 180 ggggcggaac ccccgggcgg ggccggcgcc tccccggagc tgcaactgcaccccaggctg 240 cggagcctcg ctgtgtgcgt ccaggacgtc gccccaaacc tgcagaggtgcgagcggctc 300 cccgacggcc gcgggaccta ccagtgcaag gcgaacgtct tcctgtccctgcgcgcagcc 360 gactgcctcg ccgcctga 378 102 360 DNA Mus sp. 102atggcccggt gtaggacgct ggtggccgct gccctggcgc tgctcctgcc gccagccctc 60gcgtggtaca agcccgcggc gggaccccac cactactcgg tgggccgcgc ctcggggcta 120ctgtcgagtt tccacaggtt cccgtccacg cgacgctccg agtctccagc actccgggtg 180ggaaccggac ctctgcgcaa tttagagatg cgccccagcg taaggagcct tgccctgtgt 240gtcaaagatg tgaccccgaa cctgcagagc tgccagcggc aactcaacag ccgagggact 300ttccagtgta aagcggacgt cttcttgtcg ctgcacgaga ctgattgcca gagcacctga 360103 360 DNA Rattus sp. 103 atggtccggt gtaggacgct ggtggccgcc gccctggcgctgctcctgac gccagccctc 60 gcgtggtaca agcccgcggc gggatcccac cactactcggtgggccgcgc tgcggggcta 120 ctgtcgagtt tccacaggtt cccatccacg cgacgttccgagtctccagc actccgggtg 180 ggaaccgtac ctctgcgcaa cttggagatg cgcccaagcgtaagaagcct tgccctgtgt 240 gtcaaagatg tgaccccgaa cctgcagagc tgccagcggcaactcaacag ccgagggact 300 ttccagtgta aggcggacgt cttcttgtcg ctgcacaaggctgaatgcca aagcgcctga 360 104 369 DNA Bos sp. 104 atggccgggc ccgcgatgctggtggccgcc gctctggcgc tgtgcttact gctggcgtcc 60 cctggcctcg cgtggtacaagccgacggcg gggcaggggt actactccgt gggccgcgcc 120 gcggggctgc tgtccggcttccacaggtcg ccgtacgcac ggcgctccga gccccgcggg 180 ggcacgcgat ccctgggaggggtcggcact ttccgggaga tgcgccccaa cctgcggagt 240 cttgccgtgt gcgtcgaggaggtcaccccc aacctgcaga gctgcgagcc actccccgac 300 ggccgcgcca ctttccagtgcaaggccgac gtcttcctgt cgctcagcgc ctcggactgt 360 cgcaagtga 369 105 996DNA Bos sp. 105 atgcacaacg cgtcgtactg ggggccggag cgcgccaaca cgtcgtgccccgcgcccgca 60 cccacgctcg gctgtcccaa cgcgtccggg ccggcgccgc cgctgccgccgccgctggcc 120 gtagccgtgc ccgttgtgta cgcggtgatc tgcgcagtgg gactggcgggcaactcggcg 180 gtactgttcg tgctgctgcg ggcgccgcgc aggaagaccg tcaccaacctgttcatcctc 240 aacctggccg tggccgacga gcttttcacg ctcgtgccgc ctgtcaacatcgccgacttt 300 ctgctgaggc gctggccctt cggggagctc ctatgcaagc tcgtcgtggccgtcgatcag 360 tacaacacct tctccagcct ctatttcctc acggtcatga gcgccgaccgctacctggtg 420 gtgctggcca ccgccgagtc gcgccgggtg gccggccgca cgtacggcgccgcgcgcgcg 480 gtgagcctgg ccgtctgggg ggtcgcgacc ctggtggtgc tgcccttcgcggtgttcgcg 540 cggctcgacg aggagcaggg ccggcgccag tgcgtactgg tcttcccgcagcccgaggcc 600 ttgtggtggc gcgcgagccg cctgtacacg ctggtgctcg gcttcgccatcccagtgtcc 660 accatctgcg tcctctacac ctcgctgctg tgccggctgc gcgccatacgcctcgacagc 720 cacgccaagg ccctggaccg cgccaagaag cgggtgaccg tcctggtggtggccatcctg 780 gccgtgtgcc tcctcgtctg gacgccctac cacctgagca ccgtggtggcgctcaccacc 840 gacctcccgc agacgccgct ggtcatcgcc gtgtcctact tcatcaccagcctgagctac 900 gccaacagct gcctcaaccc tttcctctac gccttcctgg acgacagcttccgccggagc 960 ctccgccagc tgctggcgtg ccgcaccacc tcctga 996 106 1011 DNABos sp. 106 atgatggagg ccactgggct ggaaggcctg gaaagcacaa gctccccctgcccaggtagc 60 acaggcaccg gcctctcatg ggacaatggc accagacaca atgccaccttccccgagccg 120 ctgcctgccc tctacgtgct gctgccggtg gtatactctg tcatctgtgccgtggggctg 180 gtgggcaacg cagccgtcat ctgtgtgatc ctgagggctc ccaagatgaagacagtgacc 240 cacgtgttca tcctgaacct ggccatcgcc gacgggctct tcacgctggtgctgcccacc 300 aatattgctg agcacctgct gcagcgctgg ccctttgggg aggtgctctgcaagctggtg 360 ctggccattg accactgcaa catcttctcc agtgtctact tcctggccgccatgagtata 420 gaccgctacc tggtggttct ggccacggca cgctcccgcc gcatgccccggcgcaccgtc 480 cacagggcga aggtcgccag cctgtgcgtc tggctgggtg tcacagtcgcagtgctgccc 540 ttccttacct tcgcaggcgt gtacaacaat gagctgcagg tcacaagttgtgggctgagt 600 ttcccgcggc ccgagagggc ctggttccag gcaagccgca tctacacgctggtgctgggc 660 ttcgtggtgc ccatgtgcac cctctgcgtg ctctacgcag acctgctgcggaggctaagg 720 gccctgcggc tccactccgg agccaaggct ctgggcaagg ccaagcggaaggttagcctc 780 ctggtcctgg ccgtgctggc cgtgggcctg ctctgctgga cgcccttccacctggcctca 840 attgtggccc tgaccacaga cctgccccag acaccgctgg tcatcatcgtctcctatgtg 900 gtcaccagcc tcagctacac cagctcctgc ctcaacccct tcctctatgccttcctggat 960 cacagcttcc ggaagagcct ccgcaccgca tgtcggtgcc agggggcata a1011 107 29 PRT Homo sapiens MOD_RES (1) D-Trp(6Br) 107 Trp Tyr Lys ProAla Ala Gly His Ser Ser Tyr Ser Val Gly Arg Ala 5 10 15 Ala Gly Leu LeuSer Gly Leu Arg Arg Ser Pro Tyr Ala 20 25 108 29 PRT Bos sp. MOD_RES (1)D-Trp(6Br) 108 Trp Tyr Lys Pro Thr Ala Gly Gln Gly Tyr Tyr Ser Val GlyArg Ala 1 5 10 15 Ala Gly Leu Leu Ser Gly Phe His Arg Ser Pro Tyr Ala 2025

1. A peptide containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:1, whichmay optionally be brominated at the N-terminal amino acid residue, orits amide or ester, or a salt thereof.
 2. The peptide or its amide orester, or a salt thereof, according to claim 1, which contains the sameor substantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:1.
 3. The peptide or its amide or ester, or asalt thereof, according to claim 1, which has the amino acid sequencerepresented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:66. 4.The peptide or its amide or ester, or a salt thereof, according to claim1, which is 6-brominated at the N-terminal tryptophan residue and hasthe amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3 or SEQ ID NO:66.
 5. A peptide containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:4, which may optionally be brominated at the N-terminal aminoacid residue, or its amide or ester, or a salt thereof.
 6. The peptideor its amide or ester, or a salt thereof, according to claim 5, whichcontains the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:4.
 7. The peptide or itsamide or ester, or a salt thereof, according to claim 5, which has theamino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6or SEQ ID NO:67.
 8. The peptide or its amide or ester, or a saltthereof, according to claim 5, which is 6-brominated at the N-terminaltryptophan residue and has the amino acid sequence represented by SEQ IDNO:67.
 9. The peptide or its amide or ester, or a salt thereof,according to claim 5, which is 6-brominated at the N-terminal tryptophanresidue and has the amino acid sequence represented by SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6 or SEQ ID NO:67.
 10. A peptide containing the sameor substantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:7, which may optionally be brominated at theN-terminal amino acid residue, or its amide or ester, or a salt thereof.11. The peptide or its amide or ester, or a salt thereof, according toclaim 10, which contains the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:7.
 12. Thepeptide or its amide or ester, or a salt thereof, according to claim 10,which has the amino acid sequence represented by SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:68 or SEQ ID NO:69.
 13. A peptide containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:13, which may optionally be brominated at theN-terminal amino acid residue, or its amide or ester, or a salt thereof.14. The peptide or its amide or ester, or a salt thereof, according toclaim 13, which contains the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:13.
 15. Thepeptide or its amide or ester, or a salt thereof, according to claim 13,which has the amino acid sequence represented by SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 or SEQ ID NO:70.
 16. A peptide containing the sameor substantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:16, which may optionally be brominated at theN-terminal amino acid residue, or its amide or ester, or a salt thereof.17. The peptide or its amide or ester, or a salt thereof, according toclaim 16, which contains the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:16.
 18. Thepeptide or its amide or ester, or a salt thereof, according to claim 17,which has the amino acid sequence represented by SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18 or SEQ ID NO:71.
 19. The peptide or its amide orester, or a salt thereof, according to claim 1, which is capable ofbinding to a protein or its salt containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:49 or SEQ ID NO:86.
 20. The peptide or its amide or ester, ora salt thereof, according to claim 1, which is capable of binding to aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:59.21. The peptide or its amide or ester, or a salt thereof, according toclaim 1, which is capable of binding to a protein or its salt containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:84 or SEQ ID NO:88.
 22. A partialpeptide of the peptide according to claim 1, or its amide or ester, or asalt thereof.
 23. A precursor peptide of the peptide according to claim1, or its amide or ester, or a salt thereof.
 24. The precursor peptideor its amide or ester, or a salt thereof, according to claim 23, whichcontains the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:19.
 25. The peptide or itsamide or ester, or a salt thereof, according to claim 24, which has theamino acid sequence represented by SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21 or SEQ ID NO:72.
 26. The peptide or its amide or ester, or a saltthereof, according to claim 23, which contains the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:22.
 27. The peptide or its amide or ester, or a salt thereof,according to claim 26, which has the amino acid sequence represented bySEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:73.
 28. Apolynucleotide containing a polynucleotide encoding the peptideaccording to claim
 1. 29. The polynucleotide according to claim 28,which has the base sequence represented by SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77,SEQ ID NO:78 or SEQ ID NO:79.
 30. A polynucleotide containing apolynucleotide encoding the partial peptide according to claim
 22. 31. Apolynucleotide containing a polynucleotide encoding the precursorpeptide according to claim
 23. 32. The polynucleotide according to claim31, which has the base sequence represented by SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:80 or SEQ ID NO:81.
 33. The polynucleotide according to any one ofclaims 28 to 32, which is a DNA.
 34. A recombinant vector containing thepolynucleotide according to any one of claims 28 to
 32. 35. Atransformant transformed with the recombinant vector according to claim34.
 36. A method of manufacturing the peptide, its partial peptide orits precursor peptide, or a salt thereof, according to claim 1, whichcomprises culturing the transformant according to claim 35 and producingthe peptide, partial peptide or precursor peptide according to claim 1.37. An antibody to the peptide, its partial peptide or its precursorpeptide, or its amide or ester, or a salt thereof, according to claim 1.38. The antibody according to claim 37, which is a neutralizing antibodyto inactivate the activity of the peptide, its partial peptide, or itsamide or ester, or a salt thereof, according to claim
 1. 39. Apharmaceutical comprising the antibody according to claim
 37. 40. Thepharmaceutical according to claim 39, which is a preventive/therapeuticagent for obesity or hyperphagia.
 41. A diagnostic product comprisingthe antibody according to claim
 37. 42. The diagnostic product accordingto claim 41, which is a diagnostic product for anorexia, obesity orhyperphagia.
 43. A pharmaceutical comprising the peptide, its partialpeptide, or its amide or ester, or a salt thereof, according to claim 1.44. The pharmaceutical according to claim 43, which is apreventive/therapeutic agent for anorexia or eating stimulant.
 45. Apharmaceutical comprising the polynucleotide according to claim
 28. 46.The pharmaceutical according to claim 45, which is apreventive/therapeutic agent for anorexia or eating stimulant.
 47. Adiagnostic product comprising the polynucleotide according to claim 28.48. The diagnostic product according to claim 47, which is a diagnosticproduct for anorexia, obesity or hyperphagia.
 49. A polynucleotidecontaining a complementary base sequence to the polynucleotide accordingto claim 28, or a part thereof.
 50. A pharmaceutical comprising thepolynucleotide according to claim
 49. 51. The pharmaceutical accordingto claim 50, which is a preventive/therapeutic agent for obesity orhyperphagia.
 52. A method of screening a compound or its salt thatalters the binding property between the peptide, its partial peptide, orits amide or ester, or a salt thereof, according to claim 1 and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:49,which comprises using the peptide, its partial peptide, or its amide orester, or a salt thereof, according to claim 1 and a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:49.
 53. A method ofscreening a compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to claim 1 and a protein or its salt containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:59, which comprises using the peptide,its partial peptide, or its amide or ester, or a salt thereof, accordingto claim 1 and a protein or its salt containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:59.
 54. A method of screening a compound or itssalt that alters the binding property between the peptide, its partialpeptide, or its amide or ester, or a salt thereof, according to claim 1and a protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:84, which comprises using the peptide, its partial peptide, or itsamide or ester, or a salt thereof, according to claim 1 and a protein orits salt containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:84.
 55. Akit for screening a compound or its salt that alters the bindingproperty between the peptide, its partial peptide, or its amide orester, or a salt thereof, according to claim 1 and a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:49, comprising the peptide,its partial peptide, or its amide or ester, or a salt thereof, accordingto claim 1 and a protein or its salt containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:49.
 56. A kit for screening a compound or itssalt that alters the binding property between the peptide, its partialpeptide, or its amide or ester, or a salt thereof, according to claim 1and a protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:59, comprising the peptide, its partial peptide, or its amide orester, or a salt thereof, according to claim 1 and a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:59.
 57. A kit for screeninga compound or its salt that alters the binding property between thepeptide, its partial peptide, or its amide or ester, or a salt thereof,according to claim 1 and a protein or its salt containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:84, comprising the peptide, its partialpeptide, or its amide or ester, or a salt thereof, according to claim 1and a protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:84.
 58. A compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to claim 1 and a protein or its salt containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:49, which is obtainable by using thescreening method according to claim 52 or the screening kit according toclaim
 55. 59. A compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to claim 1 and a protein or its salt containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:59, which is obtainable by using thescreening method according to claim 53 or the screening kit according toclaim
 56. 60. A compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to claim 1 and a protein or its salt containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:84, which is obtainable by using thescreening method according to claim 54 or the screening kit according toclaim
 57. 61. The compound or its salt according to any one of claims 58to 60, which is an agonist.
 62. The compound or its salt according toany one of claims 58 to 60, which is an antagonist.
 63. A pharmaceuticalcomprising the compound or its salt according to any one of claims 58 to60.
 64. A preventive/therapeutic agent for anorexia or eating stimulantcomprising the agonist according to claim
 61. 65. Apreventive/therapeutic agent for obesity or hyperphagia comprising theantagonist according to claim
 62. 66. An antiobesity agent obtainable byusing the screening method according to claim 52 or the screening kitaccording to claim
 55. 67. An antiobesity agent obtainable by using thescreening method according to claim 53 or the screening kit according toclaim
 56. 68. An antiobesity agent obtainable by using the screeningmethod according to claim 54 or the screening kit according to claim 57.69. A method of screening a compound or its salt that alters theexpression level of the peptide, its partial peptide or its precursorpeptide according to claim 1, which comprises using a DNA encoding thepeptide, its partial peptide or its precursor peptide according toclaim
 1. 70. A kit for screening a compound or its salt that alters theexpression level of the peptide, its partial peptide or its precursorpeptide according to of claim 1, comprising a DNA encoding the peptide,its partial peptide or its precursor peptide according to claim
 1. 71. Acompound or its salt that alters the expression level of the peptide,its partial peptide or its precursor peptide according to claim 1, whichis obtainable by using the screening method according to claim 69 or thescreening kit according to claim
 70. 72. The compound or its saltaccording to claim 71, which is a compound or its salt that increasesthe expression level.
 73. The compound or its salt according to claim71, which is a compound or its salt that decreases the expression level.74. A pharmaceutical comprising the compound or its salt according toclaim
 71. 75. A preventive/therapeutic agent for anorexia or eatingstimulant comprising the compound or its salt according to claim
 72. 76.A preventive/therapeutic agent for obesity or hyperphagia comprising thecompound or its salt according to claim
 73. 77. A method forpreventing/treating anorexia, which comprises administering to a mammalan effective amount of the peptide, its partial peptide, or its amide orester, or a salt thereof, according to claim 1, the polynucleotideaccording to claim 28, the agonist according to claim 61, or thecompound or its salt according to claim
 72. 78. A method for stimulatingappetite, which comprises administering to a mammal an effective amountof the peptide, its partial peptide, or its amide or ester, or a saltthereof, according to claim 1, the polynucleotide according to claim 28,the agonist according to claim 61, or the compound or its salt accordingto claim
 72. 79. A method for preventing/treating obesity orhyperphagia, which comprises administering to a mammal an effectiveamount of the antibody according to claim 37, the polynucleotideaccording to claim 49, the antagonist according to claim 62, or thecompound or its salt according to claim
 73. 80. A protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:86.
 81. The protein or itssalt according to claim 80, containing the amino acid sequencerepresented by SEQ ID NO:86.
 82. A partial peptide or its salt of theprotein according to claim
 80. 83. A polynucleotide containing apolynucleotide encoding the protein according to claim 80, or a partialpeptide thereof.
 84. The polynucleotide according to claim 83, which isa DNA.
 85. The polynucleotide according to claim 84, which contains thebase sequence represented by SEQ ID NO:87.
 86. A recombinant vectorcontaining the polynucleotide according to claim
 83. 87. A transformanttransformed by the recombinant vector according to claim
 86. 88. Amethod of manufacturing the protein according to claim 80, its partialpeptide or a salt thereof, which comprises culturing the transformantaccording to claim 87 and producing the protein according to claim 80,its partial peptide or a salt thereof.
 89. A pharmaceutical comprisingthe protein according to claim 80 or the partial peptide according toclaim 82, or a salt thereof.
 90. A pharmaceutical comprising thepolynucleotide according to claim
 83. 91. The pharmaceutical accordingto claim 90, which is a preventive/therapeutic agent for anorexia oreating stimulant.
 92. A diagnostic product comprising the polynucleotideaccording to claim
 83. 93. The diagnostic product according to claim 92,which is a diagnostic product for anorexia, obesity or hyperphagia. 94.An antibody to the protein according to claim 80 or the partial peptideaccording to claim 82, or a salt thereof.
 95. The antibody according toclaim 94, which is a neutralizing antibody to inactivate signaltransduction of the protein according to claim
 80. 96. A pharmaceuticalcomprising the antibody according to claim
 94. 97. The pharmaceuticalaccording to claim 96, which is a preventive/therapeutic agent forobesity or hyperphagia.
 98. A diagnostic product comprising the antibodyaccording to claim
 94. 99. The diagnostic product according to claim 98,which is a diagnostic product for anorexia, obesity or hyperphagia. 100.A polynucleotide containing a complementary base sequence to thepolynucleotide according to claim 83, or a part thereof.
 101. Apharmaceutical comprising the polynucleotide according to claim 100.102. The pharmaceutical according to claim 101, which is apreventive/therapeutic agent for obesity or hyperphagia.
 103. A proteinor its salt containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:88. 104.The protein or its salt according to claim 103, which contains the aminoacid sequence represented by SEQ ID NO:88.
 105. A partial peptide of theprotein according to claim 103, or a salt thereof.
 106. A polynucleotidecontaining a polynucleotide encoding the protein according to claim 103or a partial peptide thereof.
 107. The polynucleotide according to claim106, which is a DNA.
 108. The polynucleotide according to claim 107,which contains the base sequence represented by SEQ ID NO:89.
 109. Arecombinant vector containing the polynucleotide according to claim 108.110. A transformant transformed with the recombinant vector according toclaim
 109. 111. A method of manufacturing the protein according to claim103, its partial peptide, or a salt thereof, which comprises culturingthe transformant of claim 110 and producing the protein according toclaim 103 or its partial peptide.
 112. A pharmaceutical comprising theprotein according to claim 103 or the partial peptide according to claim105, or a salt thereof.
 113. A pharmaceutical comprising thepolynucleotide according to claim
 106. 114. The pharmaceutical accordingto claim 113, which is a preventive/therapeutic agent for anorexia oreating stimulant.
 115. A diagnostic product comprising thepolynucleotide according to claim
 106. 116. The diagnostic productaccording to claim 115, which is a diagnostic product for anorexia,obesity or hyperphagia.
 117. An antibody to the protein according toclaim 103 or the partial peptide according to claim 105, or a saltthereof.
 118. The antibody according to claim 117, which is aneutralizing antibody to inactivate signal transduction of the proteinaccording to claim
 103. 119. A pharmaceutical comprising the antibodyaccording to claim
 117. 120. The pharmaceutical according to claim 119,which is a preventive/therapeutic agent for obesity or hyperphagia. 121.A diagnostic product comprising the antibody according to claim 117.122. The diagnostic product according to claim 121, which is adiagnostic product for anorexia, obesity or hyperphagia.
 123. Apolynucleotide containing a complementary base sequence to thepolynucleotide according to claim 106, or a part thereof.
 124. Apharmaceutical comprising the polynucleotide according to claim 123.125. The pharmaceutical according to claim 124, which is apreventive/therapeutic agent for obesity or hyperphagia.
 126. A methodof screening a compound or its salt that alters the binding propertybetween the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to claim 1 and a protein or its salt containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:86, which comprises using the peptide,its partial peptide, or its amide or ester, or a salt thereof, accordingto claim 1 and a protein or its salt containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:86.
 127. A method of screening a compound orits salt that alters the binding property between the peptide, itspartial peptide, or its amide or ester, or a salt thereof, according toclaim 1 and a protein or its salt containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:88, which comprises using the peptide, its partial peptide, orits amide or ester, or a salt thereof, according to claim 1 and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:88.128. A kit for screening a compound or its salt that alters the bindingproperty between the peptide, its partial peptide, or its amide orester, or a salt thereof, according to claim 1 and a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:86, comprising the peptide,its partial peptide, or its amide or ester, or a salt thereof, accordingto claim 1 and a protein or its salt containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:86.
 129. A kit for screening a compound or itssalt that alters the binding property between the peptide, its partialpeptide, or its amide or ester, or a salt thereof, according to claim 1and a protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:88, comprising the peptide, its partial peptide, or its amide orester, or a salt thereof, according to claim 1 and a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:88.
 130. A compound or itssalt that alters the binding property between the peptide, its partialpeptide, or its amide or ester, or a salt thereof, according to claim 1and a protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:86, which is obtainable by using the screening method according toclaim 126 or the screening kit according to claim
 128. 131. A compoundor its salt that alters the binding property between the peptide, itspartial peptide, or its amide or ester, or a salt thereof, according toclaim 1 and a protein or its salt containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:88, which is obtainable by using the screening methodaccording to claim 127 or the screening kit according to claim
 129. 132.The compound or its salt according to any one of claims 130 or 131,which is an agonist.
 133. The compound or its salt according to any oneof claims 130 or 131, which is an antagonist.
 134. A pharmaceuticalcomprising the compound or its salt according to any one of claims 130or
 131. 135. A preventive/therapeutic agent for anorexia or eatingstimulant, comprising the agonist according to claim
 132. 136. Apreventive/therapeutic agent for obesity or hyperphagia, comprising theantagonist according to claim
 133. 137. A method of screening a compoundor its salt that alters the expression level of a protein containing thesame or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:86, which comprises using a DNAencoding a protein containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:86.138. A kit for screening a compound or its salt that alters theexpression level of a protein containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:86, comprising a DNA encoding a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:86.
 139. A compound or its salt that alters theexpression level of a protein containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:86, which is obtainable by using the screening method according toclaim 137 or the screening kit according to claim
 138. 140. The compoundor its salt according to claim 139, which is a compound or its salt thatincreases the expression level.
 141. The compound or its salt accordingto claim 139, which is a compound or its salt that decreases theexpression level.
 142. A pharmaceutical comprising the compound or itssalt according to claim
 139. 143. A preventive/therapeutic agent foranorexia or eating stimulant comprising the compound or its saltaccording to claim
 140. 144. A preventive/therapeutic agent for obesityor hyperphagia comprising the compound or its salt according to claim141.
 145. A method of screening a compound or its salt that alters theexpression level of a protein containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:88, which comprises using a DNA encoding a protein containing thesame or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:88.
 146. A kit for screening acompound or its salt that alters the expression level of a proteincontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:88, comprising a DNAencoding a protein containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:88.147. A compound or its salt that alters the expression level of aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:88, whichis obtainable by using the screening method according to claim 145 orthe screening kit according to claim
 146. 148. The compound or its saltaccording to claim 147, which is a compound or its salt that increasesthe expression level.
 149. The compound or its salt according to claim147, which is a compound or its salt that decreases the expressionlevel.
 150. A pharmaceutical comprising the compound or its saltaccording to claim
 147. 151. A preventive/therapeutic agent for anorexiaor eating stimulant comprising the compound or its salt according toclaim
 148. 152. A preventive/therapeutic agent for obesity orhyperphagia comprising the compound or its salt according to claim 149.153. A method for preventing/treating anorexia, which comprisesadministering to a mammal an effective amount of the protein accordingto claim 80, its partial peptide or a salt thereof, the polynucleotideaccording to claim 83, the protein according to claim 103, its partialpeptide or a salt thereof, the polynucleotide according to claim 106,the agonist according to claim 132, the compound or its salt accordingto claim 140, or the compound or its salt according to claim
 148. 154. Amethod for stimulating appetite, which comprises administering to amammal an effective amount of the protein according to claim 80, itspartial peptide or a salt thereof, the polynucleotide according to claim83, the protein according to claim 103, its partial peptide or a saltthereof, the polynucleotide according to claim 106, the agonistaccording to claim 132, the compound or its salt according to claim 140,or the compound or its salt according to claim
 148. 155. A method forpreventing/treating obesity or hyperphagia, which comprisesadministering to a mammal an effective amount of the antibody accordingto claim 94, the polynucleotide according to claim 100, the antibodyaccording to claim 117, the polynucleotide according to claim 123, theantagonist according to claim 133, the compound or its salt according toclaim 141, or the compound or its salt according to claim
 149. 156. Anon-human mammal bearing the DNA according to claim 28, which isexogenous, or its variant DNA.
 157. The mammal according to claim 156,wherein the non-human mammal is a rodent.
 158. A recombinant vectorbearing the exogenous DNA or its variant DNA according to claim 28 andcapable of expressing in a mammal.
 159. A non-human embryonic stem cell,wherein the DNA according to claim 28 is inactivated.
 160. The embryonicstem cell according to claim 159, wherein the DNA is inactivated byintroducing a reporter gene.
 161. The embryonic stem cell according toclaim 159, wherein the non-human mammal is a rodent.
 162. A non-humanmammal deficient in expressing the DNA according to claim 28, whereinthe DNA is inactivated.
 163. A non-human mammal according to claim 162,wherein the DNA is inactivated by inserting a reporter gene therein andthe reporter gene is capable of expressing under control of a promoterfor the DNA according to claim
 28. 164. The non-human mammal accordingto claim 162, which is a rodent.
 165. A method of screening a compoundor its salt that promotes or inhibits the activity of a promoter for theDNA according to claim 28, which comprises administering a test compoundto the mammal according to claim 163 and detecting expression of thereporter gene.
 166. A non-human mammal bearing the DNA according toclaim 83, which is exogenous, or its variant DNA.
 167. The mammalaccording to claim 166, wherein the non-human mammal is a rodent.
 168. Arecombinant vector bearing the exogenous DNA or its variant DNAaccording to claim 83 and capable of expressing in a mammal.
 169. Anon-human embryonic stem cell, wherein the DNA according to claim 83 isinactivated.
 170. The embryonic stem cell according to claim 169,wherein the DNA is inactivated by introducing a reporter gene.
 171. Theembryonic stem cell according to claim 169, wherein the non-human mammalis a rodent.
 172. A non-human mammal deficient in expressing the DNAaccording to claim 83, wherein the DNA is inactivated.
 173. A non-humanmammal according to claim 172, wherein the DNA is inactivated byinserting a reporter gene therein and the reporter gene is capable ofexpressing under control of a promoter for the DNA according to claim83.
 174. The non-human mammal according to claim 172, which is a rodent.175. A method of screening a compound or its salt that promotes orinhibits the activity of a promoter for the DNA according to claim 83,which comprises administering a test compound to the mammal according toclaim 173 and detecting expression of the reporter gene.
 176. Anon-human mammal bearing the DNA according to claim 106, which isexogenous, or its variant DNA.
 177. The mammal according to claim 176,wherein the non-human mammal is a rodent.
 178. A recombinant vectorbearing the exogenous DNA according to claim 106 or its variant DNA andcapable of expressing in a mammal.
 179. A non-human embryonic stem cell,wherein the DNA according to claim 106 is inactivated.
 180. Theembryonic stem cell according to claim 179, wherein the DNA isinactivated by introducing a reporter gene.
 181. The embryonic stem cellaccording to claim 179, wherein the non-human mammal is a rodent.
 182. Anon-human mammal deficient in expressing the DNA according to claim 106,wherein the DNA is inactivated.
 183. The non-human mammal according toclaim 182, wherein the DNA is inactivated by inserting a reporter genetherein and the reporter gene is capable of expressing under control ofa promoter for the DNA according to claim
 106. 184. The non-human mammalaccording to claim 182, which is a rodent.
 185. A method of screening acompound or its salt that promotes or inhibits the activity of apromoter for the DNA according to claim 106, which comprisesadministering a test compound to the mammal according to claim 183 anddetecting expression of the reporter gene.
 186. Use of the peptide, itspartial peptide, its amide or ester, or a salt thereof, according toclaim 1, the polynucleotide according to claim 28, the agonist accordingto claim 61, or the compound or its salt according to claim 72, formanufacturing a preventive/therapeutic agent for anorexia.
 187. Use ofthe peptide, its partial peptide, its amide or ester, or a salt thereof,according to claim 1, the polynucleotide according to claim 28, theagonist according to claim 61, or the compound or its salt according toclaim 72, for manufacturing an eating stimulant.
 188. Use of theantibody according to claim 37, the polynucleotide according to claim49, the antagonist according to claim 62, or the compound or its saltaccording to claim 73, for manufacturing a preventive/therapeutic agentfor obesity or hyperphagia.
 189. Use of the protein according to claim80 or its partial peptide or a salt thereof, the polynucleotideaccording to claim 83, the protein according to claim 103 or its partialpeptide or a salt thereof, the polynucleotide according to claim 106,the agonist according to claim 132, the compound or its salt accordingto claim 140 or the compound or its salt according to claim 148, formanufacturing a preventive/therapeutic agent for anorexia.
 190. Use ofthe protein according to claim 80 or its partial peptide or a saltthereof, the polynucleotide according to claim 83, the protein accordingto claim 103 or its partial peptide or a salt thereof, thepolynucleotide according to claim 106, the agonist according to claim132, the compound or its salt according to claim 140 or the compound orits salt according to claim 148, for manufacturing an eating stimulant.191. Use of the antibody according to claim 94, the polynucleotideaccording to claim 100, the antibody according to claim 117, thepolynucleotide according to claim 123, the antagonist according to claim133, the compound or its salt according to claim 141, or the compound orits salt according to claim 149, for manufacturing apreventive/therapeutic agent for obesity or hyperphagia.